Gene editing our way to a precision medicine future: novel KRAS models of pancreatic cancer

 

Published! is a series of chats with women in science highlighting their publications as leading authors. If you know someone who has recently published a scientific manuscript who would like to chat with me, you can get in touch via Twitter @laurenpoppi or email <lauren.poppi@rutgers.edu>.

Dr Maria De La Paz Zafra Martin is a cancer biology postdoctoral fellow at Weill Cornell University in New York, who recently published an article in Cancer Discovery. Maria has spent the last few years developing new models of pancreatic cancer using cutting-edge gene editing technology. The models are based on mutations in a gene called ‘KRAS’ from human cancer patients. Maria tells us a bit about gene editing and its limitations, precision medicine, why it is important to stay adaptive as a scientist, and the importance of finding a happy and collaborative lab environment.
You can read Maria and the Dow lab’s article in Cancer Discovery here.

Congratulations on your publication, Maria! Could you tell us a bit about your findings?
Basically, we used CRISPR to create new mouse models in the cancer field. When I joined the lab back in 2015, CRISPR was a very new and exciting tool. It allows us to introduce mutations in a very easy manner. We used this technology to develop new mouse models that are based on mutations in the gene KRAS that we were finding in cancer patients. I addressed questions in pancreatic cancer, because KRAS mutations are very important there, and we had done an initial study on tumour initiation with the new mutations that we created that led us in that direction. It is not only important to look at the mutation itself but also what type of alteration can happen in that gene, because based on that it can have a different therapeutic target.

You were able to show that cancer cells carrying certain mutations were more sensitive to certain types of treatments. That’s so cool and very translational! 
Yeah! Of course, we love biology and we are very interested in finding biological differences, but my goal is really to exploit those differences to target cancer therapeutically. 

So, in theory, we could biopsy a tumour, work out what mutations are present, then based on that decide which treatment will be best for that person’s tumour?
Yes, it’s what we call ‘precision medicine’ and I think it is where we are going. We have to stop thinking about cancer in terms of ‘pancreatic cancer’ versus ‘colorectal cancer’. Tumours can be so different depending on the patient, we were overlooking this before. Now that we have next generation sequencing, we can get a lot of information from tumours, including their mutational status, as well as copy number alterations. There is a lot of drug development happening in parallel, so I think we are converging on that same idea of precision medicine. One patient gets one very specific treatment. That’s the future.

Would you be able to briefly describe how CRISPR works?
At least in the work that we are talking about today, CRISPR allows us to manipulate the DNA of cells to change one base pair, resulting in a change of amino acid. We created, in this case, an activating mutation. CRISPR is like molecular scissors that find a specific area in the DNA, with help from a guide, and it cuts the DNA in that area. I also provide a template to ‘repair’ that cut, in a way. But in that repair template, I introduced a mutation. You can introduce whatever you want, as long as the ‘arms’ of the template are similar to the surrounding DNA where you want to incorporate it. The cell does everything else for you! Because the cell is very stressed, like “Oh my god! Cas9 just cut here!”, it is going to go crazy trying to repair the DNA. It will just take the template we provided and introduce it, thinking that it’s the proper DNA. 

Do you think we are going to start CRISPR-ing people?
Yes and no. For example, cancer is a very complex disease, you have multiple mutations… so I think it’s going to be impossible to cure cancer with CRISPR. But sometimes you have only one mutation, like in the case of a rare disease, and then you can repair it, using other gene editing technologies, like base editing. They’re using adenovirus to deliver the base editors and correct the point mutation. I think that we are still a long way off from doing this. Everything needs to be extremely regulated. We need to go step by step. We know that gene editing doesn’t always work perfectly. Sometimes it does what you are hoping it is going to do, like introduce a mutation or a repair, but sometimes it has off-target effects as well. We have to be very careful with it!

Were there any unexpected findings that cropped up along the way in this project?
The unexpected thing occurred at the very beginning actually (laughs). When I joined the lab, my PI said, “This will be very straightforward. We have already started the cells, so we will make the mice, and we’re going to see differences for sure Maria. Six months”. So, I started the cells in vitro. Our lab studies colorectal cancer, so the cells I was using expressed colon genes. At the same time, CRISPR technology was still being developed, so we were struggling with how to cleanly apply the technology. As I was saying before, CRISPR can have off-target effects. We needed to introduce the mutation without touching anything else. In the beginning, we were introducing the mutation but we were also altering a lot of other stuff. It took me two years of targeting, and changing my strategies of targeting, and changing the stem cells to be successful. That’s actually why I moved to pancreatic cancer. We had to change the cell line because every time we changed the targeting strategy, we would have another issue. 

Oh my gosh!
Yeah. So the idea that we would have our first mouse model in six months… it took us two and a half years to get there! At some point I thought, “I don’t know if this is gonna’ work”. I want to say… keep trying. CRISPR was still a new technology, so papers were coming out all the time. There was a new Cas9 variant that had less off-target effects, and once we started using that particular variant we were able to create the mutation cleanly. Two and a half years!

Typical of science though isn’t it? If anyone ever says, “We will be done with this in six months”, alarm bells start ringing (laughs). Were there any more challenges you had to face?
That was the main one. It’s important to keep adapting. Once we finally got the mice, the mutation was in a different organ that I didn’t have any expertise in. This was another challenge because I had to study KRAS in the context of pancreatic cancer. My PI Luke (A/Prof. Lukas Dow) said to me, “Maria, we study colorectal cancer… you figure it out!” (laughs). I was like, “OK!”. This is why I like science. You need a background in biology and in general lab techniques, but you don’t know what is going to come next. You don’t know where your project is going to take you. You have to be ready to go in that direction, like, “Let’s go! Let’s do this!”.

How was the writing process?
I actually really liked it. I’m always very nervous at the beginning, when you have that blank page. You open the document and close it, then open it, and close it, and nothing happens for a while. I always start with methods. I start with things that I know, which makes me comfortable writing, and then I can start tackling Figure 1, for example. It’s a process, and it’s actually fun. Luke is really helpful and is always providing feedback, it’s a very dynamic approach and I like it. 

What are the next questions that you and your lab want to answer?
One good thing is that you can now cross these different KRAS models to different organs. A lot of people in other labs and in our lab are addressing questions in other cancers. But now I really am interested in pancreatic cancer. We talked about tumour initiation in this paper and how it’s different. Now I’m interested in how these different KRAS mutations play a role in tumour progression and within the context of therapy. I had very preliminary data in organoids, but of course I want to do it in vivo. I think I’m going to stay in the pancreatic cancer field for now – if I don’t have another adventure – and I’m going to try to better understand how these mutations work.  

Do you remember a specific moment that you realized you wanted to be a scientist, or was it a gradual progression?
I guess it was the second. I’m a biochemist, so when I was about to finish my degree I thought, “OK what’s next?”. I started doing research, and I liked it. I don’t have an impressive story to tell. You’re around other people and they’re doing the same thing. I didn’t want to go to industry, that’s for sure. But I wasn’t sure about my next step. I always thought if I didn’t like it, I would quit, but I’m still here (laughs)!

Do you have a fun science fact that you would like to share with us?
The naked mole rat! I didn’t know about them back in Spain, but I wasn’t working in cancer then. But then I got here, and everyone was like, “Have you ever seen a naked mole rat?”. I looked it up - and it’s the most disgusting animal in the world (laughs) - but they live very long lives and never develop cancer. So they make a very good model. There are a lot of papers coming out now on naked mole rats. Two years ago for Halloween, I dressed up as one (laughs)!

That’s the best (laughs)!
I was so obsessed. I had the teeth, I could barely speak. So every time I wanted to say something I had to take the teeth out first (laughs). I’ll have to send you a photo.

Science is a stressful job! What do you like to do to destress? 
Honestly, I go on a hike. I need to start doing that again. Hiking, climbing sometimes. I also just like to go out with friends, talk to them, have dinner. Nothing special! I don’t have a specific thing like running that I really need to do. I love living in the city, but I also like to get out on the weekends… But everyone likes to travel!

What are your thoughts on work culture in America, especially within academic research?
People work really hard here. It’s a 24/7 kind of situation. What are the good things about it? You’re in one of the best institutions so you’re probably going to be really well connected, and if you’re working 24/7 you will publish a good paper, for sure. In Spain if you work 24/7, it’s not so straight forward, it doesn’t translate directly to a good CV. Here? Most of the time it does, but it’s a really hectic life. I think it is up to you when you want to stop, or when you want to keep going. It’s up to you to take care of your mental health, and to go, “OK, let’s stop, breathe, and see what I have to change”. It’s very easy to get stuck on the hamster wheel. 

What would be your advice to a junior person wanting to be a cancer researcher such as yourself?
I’m not saying you have to love it from the very beginning, but you have to show interest. You have to be ready to learn, and ready to fail. You don’t need to know everything from the beginning. I think it is way more important to have curiosity. I was working on allergies throughout my PhD, then I decided to switch to CRISPR and cancer… so if you have the motivation, you can do whatever you want. It’s also very important to choose a lab with a good atmosphere. When you’re staying late in the lab, but your colleagues are there doing experiments too, and you’re all having fun, that helps. It’s going to be a difficult journey. It’s good to be surrounded by people that are going to support you and make your day easier. It’s really good to laugh every day! Those would be the two things I would say: stay motivated and choose a nice environment. 

What is it like being a postdoc in New York City?
It’s cool. You have to work hard, but you can play hard too! There are so many things you can do in New York. You get to meet so many people from so many different countries. Every day, you’re going to hear a random fact about a random country. There are so many things to do, concerts, free stuff. It’s the experience of a lifetime. 

Finally, something that is really relevant to the mission of GWIS… how do you hope to see the scientific landscape change for women-identifying scientists going forward, especially within your field?
We are still getting there. I think it’s a matter of time. I think there is starting to be a network that supports us. When there are more of us in this space, it’s going to be better, because we have different ways of looking at things, and it will enrich academia overall. More women involved in experiments, or tackling a problem, or even just within conversations, will change the landscape and for the better.

Do you have anything else you would like to add?
Of course! This work is not just about me, we have a very interactive and supportive lab. This work came about because of us. Thanks to Luke, and to every single member of my lab – not only because they performed experiments, but maybe because they just made happy and got me through the day! We have a cool atmosphere. Thanks to our collaborators - all of the people who were ready to help and without asking for anything in return. I love that about science in general, the collaboration. 

Read another highlight on this research, written by Ellen P. Neff.

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