The joint project began when a nine-year-old boy came to Prof. Shoshana Greenberger’s clinic at Sheba’s Safra Children’s Hospital with severe shortness of breath and was diagnosed with KLA. Seeking to deepen understanding of the disease, Greenberger approached Prof. Karina Yaniv, of Weizmann’s Immunology and Regenerative Biology Department, who for over two decades has been studying how blood and lymphatic vessels form, using zebrafish models.
In KLA, lymphatic vessels become abnormally enlarged and distorted, which keeps the lymphatic system from properly doing its job: draining fluid from tissues and supporting many essential bodily functions. As in the case of the boy treated by Greenberger, what typically brings young patients to the doctor is difficulty breathing caused by fluid buildup in the chest, but the disease also affects the skin and numerous other organs
“It was an amazing moment. Just by looking at these mutant embryos, I knew we were on the right track”
In earlier work led by Greenberger, her team at Sheba had traced the disease to a single mutation in a gene called NRAS, known to act as an oncogene. Physicians around the world started treating patients with certain cancer drugs that block NRAS or its partners, but these drugs are not always sufficiently effective and they come with harsh side effects. They failed to save the life of Greenberger’s patient, but the boy’s cells became the basis of research into the mechanisms of KLA.
“We wanted to be sure the mutation we had found really causes KLA and learn how it does that, in the hope of finding a better therapy,” says Greenberger, who heads the Multidisciplinary Center for Vascular Anomalies at Sheba. “That’s what led us to the collaboration with Weizmann.”
Zebrafish became powerful allies in this research not only because their embryos are transparent and develop rapidly, but also because their lymphatic systems share a surprising number of features with those of humans, from genetics to anatomy. The project – jointly spearheaded by Greenberger and Yaniv and led by Dr. Ivan Bassi, a postdoctoral fellow in Yaniv’s lab – began with creating a zebrafish model of the human disease. This model was initially validated by Amani Jabali, an MSc student supervised by Greenberger and Yaniv.
Since the human NRAS gene is about 80 percent identical to the zebrafish version and activates similar biochemical pathways, the researchers were able to insert the mutated human gene, taken from the cells of Greenberger’s patient, into tiny zebrafish embryos. The challenge was to ensure that the mutated gene was expressed in the lymphatic vessels alone, as it is in human disease, and nowhere else in the body. Once this was achieved, the embryos developed lymphatic abnormalities that bore a remarkable resemblance to those of human patients.
“It was an amazing moment,” Yaniv recalls. “Just by looking at these mutant embryos, I knew we were on the right track.”
The main lymphatic vessel of the embryos became grossly distorted, causing their hearts to become dilated and balloon. Further examination confirmed that these embryos shared key features with human KLA patients, including enlarged lymphatic vessels and swelling around the heart.
Using their model, Bassi and colleagues deciphered previously unknown aspects of the disease mechanism. In healthy cells, NRAS triggers cell division only when activated by a signal. In KLA, the mutated NRAS is stuck in the “on” position, causing lymphatic cells to divide and grow uncontrollably.
Hooked on discovery
The next critical step was finding a small molecule that could block the effects of the disease-causing mutation. Zebrafish embryos were perfect for this task because they enabled the testing of potential drugs on a living organism, not just isolated proteins or cells. But they also posed a major headache. High-throughput screening, the standard method for quickly testing large numbers of compounds, is normally automated. The tricky part was figuring out how to get each zebrafish into exactly the right position under a microscope – without doing it by hand – so that a machine could capture consistent images and assess the effects of treatments.
Working with collaborators, the team designed a clever automated system, in which each embryo was gently dropped into a precisely fitting slit under the microscope, where it was photographed. Then, an AI-based algorithm outlined the entire body of the larval fish and measured its area after exposure to each drug. Because the mutant fish had enlarged hearts, their total body area was significantly greater than normal, an effect expected to decrease after treatment with an effective drug.
Using this setup, the team screened about 150 small molecules, all of them existing drugs already approved for other uses. About 30 showed promising effects; two top candidates were ultimately selected through further testing.
Both these drugs reversed the KLA-like symptoms in the zebrafish model: The ballooned heart and main lymphatic vessel shrank back to their normal size and shape. To test whether these drugs might help treat the human disease, the Sheba team applied them to lymphatic cells from Greenberger’s KLA patient. The two compounds had a striking effect, blocking the cells’ abnormal sprouting, a hallmark of the disease. Importantly, both drugs have a better safety profile than the cancer drugs physicians use today to treat KLA, meaning they could cause fewer side effects.
“We hope a clinical trial will be launched soon to evaluate these drugs in patients,” Greenberger says. “Since KLA is a rare disease, we will work toward creating a multi-center collaboration to bring together enough participants.”
Meanwhile, Yaniv’s lab is using zebrafish models to explore other lymphatic disorders and to further investigate KLA. One question still puzzles them: Why does the NRAS mutation severely damage lymphatic vessels but leave veins and arteries untouched? Solving this mystery could lead to entirely new therapeutic strategies.
“These are longer-term questions,” Yaniv says, “but what we’ve found in the present study could help patients much sooner. Since the drugs we identified are already approved, getting them repurposed for KLA could move much faster than starting from scratch.”
Reference: Bassi I, Jabali A, Levin L, et al. A high-throughput zebrafish screen identifies novel candidate treatments for kaposiform lymphangiomatosis (KLA). J Exp Med. 2025;222(11):e20240513. doi: 10.1084/jem.20240513
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