From penicillin to CRISPR, the greatest scientific discoveries often have the humblest origin stories. Who knew that a little fungal contamination or a bacterial defense system would revolutionize human health and medicine? Pau Castel, a cancer researcher at New York University, has experienced this serendipity in his own lab.

When Castel joined José Baselga’s cancer research laboratory at Memorial Sloan Kettering Cancer Center for his PhD training, he was eager to work on developing targeted cancer therapies, but he reasoned that investigating the basic mechanisms driving cancer would likely be just as important.

“On one side, you have that impact by developing these drugs. But then, on the other side, you're still studying very basic mechanisms, and you can still make new discoveries on how these pathways regulate cellular behavior,” said Castel.

When Castel began his graduate research, Baselga was interested in developing therapies that inhibited phosphoinositide 3-kinases (PI3 kinases). PI3 kinases are involved in normal cellular behaviors like growth, division, and proliferation. Because of this, mutations in these enzymes commonly lead to cancer. But when Castel set out to study these mutations, he had no idea that a little more than six years later, he would discover the mechanism driving a rare vascular disease called venous malformations and complete a phase 1 clinical trial of the first ever treatment for the disease.

How did you begin studying PI3 kinase mutations?

My PhD advisor was developing clinical trials to treat cancers caused by PI3 kinase mutations, and we were trying to understand which patients would respond to those therapies and why. I thought that our models weren’t ideal for studying PI3 kinase in vivo. We based a lot of our work on cell lines and patient-derived xenografts. With mouse models, we would be able to see how the tumors initiated, whether we could use PI3 kinase inhibitors to prevent those tumors, and whether we could target the early lesions. I started this personal project to figure out how we could make mouse models of different PI3 kinase mutated cancer.

How did you create your PI3 kinase mouse models?

I conditionally activated the most common PI3 kinase mutations in different mouse tissues. We took one mouse model that was supposed to express the conditional mutation in the uterine wall because PI3 kinase is very commonly mutated in endometrial cancer. Surprisingly, those mice started getting hindlimb paralysis, and we didn’t really know why.

We said, ‘This model sucks.’ I was ready to close that project, but then I had the idea to take a different approach. 
– Pau Castel, New York University 

When we looked at these mice more closely, we found that they had what looked like vascular lesions in their spines. These lesions basically compressed the spine so the mice could not walk. We found that this uterine-expressing mouse strain was what we call “a bit leaky,” meaning that the PI3 kinase mutation was expressed not only in the uterine epithelial cells but also in a few endothelial cells. In the endothelial cells, the mutations caused vascular lesions, so we said, “This model sucks.” I was ready to close that project, but then I had the idea to take a different approach. Instead of going tissue by tissue, we took an adult mouse and used an inducible system to express a PI3 kinase mutation everywhere. Our scientific question was if we activate this everywhere, what tumor is most likely to arise in these mice? Is it breast, uterine, bladder? The answer was none of them.

What happened to the mice?

Within a few weeks of inducing the PI3 kinase mutation, these mice developed very ugly and aggressive vascular lesions. That's when we said, “Wait a second, they’re like that mouse that we thought was not specific! They might actually hold some interesting information.” Here is where all the planets aligned because my advisor’s sister, Eulàlia Baselga Torres at Sant Joan de Déu Barcelona Children's Hospital, was actually a specialist on vascular lesions in dermatology. She noticed that the lesions looked blue and that there was some blood inside of them. The lesions were a classic presentation of a disease called venous malformation.

Venous malformations are novel cavities that form in blood vessels. The cell tries to create new vessels, but it does it in a very messy way. The blood inside venous malformations is not functional because the cavity is not really connected to anything else. The blood just moves slowly, and sometimes there are not even white blood cells present. Venous malformations can cause swelling and intense pain. We had inadvertently created the very first mouse model for this disease.

What did you do with this new mouse model?

The first thing we did was to test whether we could treat the mice with PI3 kinase inhibitors. We had a lot of inhibitors in our fridge because we were studying them for cancer treatment. We treated the mice, and they responded very well. Baselga Torres then gave us samples from patients with venous malformations, and we sequenced them. To our surprise, we found that about 25 percent of them had exactly the same mutations that we see in PI3 kinase mutated cancers. All of a sudden, we went from a project that we thought was going nowhere to having the first mouse model of a rare disease, discovering PI3 kinase mutations in that disease, and showing that PI3 kinase inhibitors actually work very well to treat this disease.

All of a sudden, we went from a project that we thought was going nowhere to having the first mouse model a rare disease, discovering PI3 kinase mutations in that disease, and showing that PI3 kinase inhibitors actually work very well to treat this disease. 
– Pau Castel, New York University

While working on that project, we learned that there was another team at University College London getting similar results with a similar mouse model. We shared data and ended up publishing our findings side by side in the same issue of Science Translational Medicine (1,2). Since then, many other groups have identified these mutations in their patient cohorts.

Could PI3 kinase inhibitors treat people with venous malformations?

Because we were working with PI3 kinase inhibitors in the clinic, we already knew that they have some side effects. PI3 kinase is essential for glucose homeostasis, so these inhibitors can cause hyperglycemia and hyperinsulinemia. We reasoned that venous malformations are located just under the skin in most cases, so we thought, why not develop PI3 kinase inhibitors that we could administer topically, avoiding any side effects of systemic delivery.

As a proof-of-concept experiment, we took a cream and mixed our inhibitors into it. Despite not being optimized for our specific inhibitor, it reduced the lesion size and stopped lesion growth in the mice (1). That was the first evidence that we could administer PI3 kinase inhibitors through the skin, and it led to the founding of our company, Venthera, with the goal to develop topical PI3 kinase inhibitors to treat these patients.

What were some of the challenges of developing this drug for venous malformations?

Almost all of the PI3 kinase inhibitors were designed to be orally available. When designing an oral drug, people are interested in solubility, but to deliver a drug through the skin, we had to consider different properties such as lipophilicity and melting point. Size is also important. We cannot pass a 600 or 700 Dalton molecule through the skin, but reducing the size of a kinase inhibitor also reduces its specificity.

We went through multiple patent applications and papers to find some synthesis intermediates of PI3 kinase inhibitors that were smaller but still selective enough. We must have looked through thousands of compounds, and luckily we found one. We did some medicinal chemistry on it, which improved its selectivity and potency. We also modified it to add more lipophilic groups, and that increased its transdermal delivery but reduced its potency.

The Venthera team then came up with a clever idea to improve the molecule by turning it into a prodrug. They did this by taking advantage of esterases, which cleave ester bonds. Esterases are the most common enzymes in the skin, and they metabolize everything that goes through it. The Venthera scientists modified the lipophilic groups on our molecule with ester bonds. Like a Trojan horse, the molecule moves easily through the skin due to its lipophilic groups. Esterases cleave the ester bonds in those groups. Once through the skin, the cleaved molecule releases the PI3 kinase inhibitor. It's a beautiful pharmaceutical compound, not only because it's one of the first targeted therapies for the skin, but also because of the elegance of the approach of going from a prodrug to a drug.

Venthera recently tested this drug in a phase 1 clinical trial. How did it feel to see the positive tolerability data?

I was already happy the moment we treated a patient for the first time, but I was also extremely nervous for the possibility that there might be a negative reaction. It's not as easy as a binary happy or unhappy situation. We saw that most patients, especially at low doses, seemed to tolerate the drug well. A couple of patients developed a rash, and that is why we do a phase one dose escalation trial: to find the maximum tolerated dose. We found doses in which this rash might become more common and doses in which it's very uncommon. That helped us determine the best dose for moving forward.

What’s next for Venthera?

We’re currently looking for partners to help us develop a phase two trial. Looking further into the future, we want to develop additional ways of administering this drug for cases that can’t be treated topically. Novartis recently got approval to use their breast cancer-treating PI3 kinase inhibitor at a lower dose to treat a syndrome that causes the overgrowth of vascular anomalies. People who are taking this low dose systemic treatment are already seeing an effect on their vascular lesions. As with any drug, different pharmaceutical forms should exist, and we hope that we can make those for people with venous malformations.

Overall, how does it feel to have made a discovery in the lab and see it become a drug in clinical trials?

This has definitely been the most exciting project I’ve worked on, especially in terms of innovation, and it all started with a mistake because we were trying to do basic genetics in mice. I hope the story of this discovery and the work Venthera is doing inspire young people. My message would be to persevere because all of a sudden, a discovery may become something that's really useful to society.

This interview has been condensed and edited for clarity.

References

1. Castel, P. et al. Somatic PIK3CA mutations as a driver of sporadic venous malformations. Sci Transl Med  8, 332ra42 (2016).
2. Castillo, S.D. et al. Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans. Sci Transl Med  8, 332ra43 (2016).