Researcher Spotlight Women in Single-Cell Series
This past week, Linde Miles from the Memorial Sloan Kettering Cancer Center in New York gave an exciting talk as part of the Women in Single Cell webinar series. Linde has participated in the largest study ever to investigate the genetic roots of leukemia at the single-cell level. Last October, she co-authored a paper in Nature entitled, “Single Cell Mutation Analysis of Clonal Evolution in Myeloid Malignancies.” We were happy to sit down with Linde to discuss her career and recent work on AML.
Brittany Enzmann: Currently you’re working at the Memorial Sloan Kettering Cancer Center studying AML and other heme malignancies. Can you tell us about what brought you there?
Linde Miles: I actually came to Memorial as a graduate student. My mentor from Johns Hopkins University was moving here to be the chief of thoracic oncology and I was studying small cell lung cancer and being here, it’s a wonderful collaborative environment. You get to know a lot of the researchers and PIs, and I had met Ross Levine and had heard him speak. And at the same time, I had always been very interested in learning the differences between solid tumors and liquid tumors, like acute myeloid leukemia (AML) and other blood cancers.
When I was looking for a postdoc position, Ross was one of the very first people I contacted to discuss a postdoc position. After meeting with him and really getting to know the leukemia field a lot better and learning all the tools and tricks that they have to study the malignancy, it was a no-brainer for me to join. And they have a lot of really awesome tools and techniques that solid tumors are still trying to work out. And so learning both sides I thought would be important for my career.
Brittany: We know that many women in science face unique challenges, based on your experiences are you hopeful about the future for women in STEM?
Linde: Absolutely. I think the pandemic has specifically brought to light a lot of challenges that are specific for women, and women in STEM particularly. Women who have families at home or feel like they have obligations outside of work. And I think that those really came to light during the pandemic with a lot of people having to work from home.
I think for the most part there’s been a huge acknowledgment across the STEM field that women do have unique challenges and face unique questions and decisions that others might not. And so people are starting to be more understanding when it comes to timelines for careers and how much pressure they’re putting on the women in their labs or in their departments. And so I’m really hopeful as I start to go on the job market that we’re going to start to see a lot more understanding and empathy towards not just women, but also underrepresented minorities and different groups across the spectrum. I think there is going to be more inclusive and equitable decision-making and understanding across the board.
Brittany: Let’s jump a bit into your research. In your recent Nature publication, you investigated AML at the single-cell level. Why was looking at individual cells so important to understand this disease?
Linde: With AML patients here at MSK, everyone that comes in the door is sequenced by bulk sequencing. And bulk sequencing is super important as it really lets us know what mutations are frequently mutated in certain cancers. And those differ from cancer to cancer — what mutations seem to happen in the same patient, and are recurrent with other mutations. There has also been a lot of work that focuses on therapy response — if a patient has a certain mutation, can we put them on a specific therapy, or are they going to respond a certain way to a therapy? But the pieces that bulk sequencing doesn’t give us are regarding co-mutations. Are two mutations that frequently co-occur in the patient in the same cell? Are they synergistically cooperating and creating this really advantageous environment for that clone to grow and dominate a patient’s leukemia?
Another big question is regarding mutation order: knowing from the start how a patient’s leukemia develops. What was that initiating step that really started the train moving to leukemia? Those questions are very hard to answer with bulk sequencing and usually cannot be completely delineated just by having bulk molecular profiling done. And so we were really interested in trying to find a way to look at the single-cell resolution of a patient’s leukemia and that’s where Mission Bio came in.
Another reason that we got very involved with single-cell at a very early stage was we do a lot of mouse modeling of leukemia. That’s one of the Levine lab’s biggest strengths is the way that we model disease. We want as much information as possible about how leukemia develops and what mutations are really working together so that we can create the most accurate models of disease. And so having the resolution and knowledge at the single-cell level — to be able to say that these two mutations are in the same cell and they’re dominating a patient’s leukemia if they occur together — tells us that that’s the two mutations that we should be modeling together in a mouse and really to regenerate or recapitulate in the most accurate manner, a patient’s leukemia.
And so our use of single-cell technology was two-fold. We wanted more information about what was happening in patients but also wanted that information so that we could really make sure that our mouse modeling efforts are at the top of the game and making sure that we’re ultimately modeling something that is happening in a patient so that we can figure out a way to either prevent it or treat it.
Brittany: Thank you for going through that. You also conducted multi-omics in your study in which you looked at the genotype and immunophenotype in the same cells. Why was this important and what insights did it reveal?
Linde: When we started this study, that wasn’t something that we were really thinking about yet. And I think, as we started to get the data, it just opened up a huge range of questions. We always joke that we published all of the data, but really only talked about a fraction of it. And it opened up a ton of questions, probably more questions than we answered. For instance, when a cell picks up a mutation— whether that’s the first mutation or the third mutation that’s happening within that cell over a patient’s lifetime — what changes are happening? And are there specific alterations on the cell surface that we can see and that we can connect to a cell, picking up a mutation?
“Being able to analyze a patient’s sample and saying, ‘We know that this patient picked up a mutation A because we’re now seeing a certain protein that’s expressed on the cell surface.’ And that has so much power, not only for knowledge of how these mutations are affecting cells in particular, but there are now targeted therapeutics to certain proteins that are expressed on cells, like a CAR T cell or an antibody therapy.”
And those have shown a lot of promise in certain cancers. And I think there’s a potential that it’s something that could be used here if we can say as a cell picks up this mutation, it starting to express a protein that it didn’t express before. And it’s starting to express a protein that’s unique to that specific cell.
And having that knowledge and being able to really figure that out for some of the more frequent combinations of mutations is really important. And so, we just started scratching the surface and seeing how certain mutations affected the immunophenotype, or the cell surface proteins. The biggest change we saw was in signaling mutations. These are mutations that are thought to occur later in a patient’s disease development. These are mutations like NRAS, KRAS, or FLT3. And they are thought to be more of the oncogenic cell proliferative push that a lot of leukemias end up picking up — at the point when they’re diagnosed or have become refractory to therapy.
And those mutations actually have pretty big changes to the immunophenotype of cells that acquire them. Specifically, we saw that RAS mutations seem to induce clones to express higher levels of a protein called CD11b. And this is a more mature lineage marker, whereas cells that picked up FLT3 mutations actually seemed to express more immature immunophenotype markers. And so the fact that two oncogenic mutations that are in the same class ( in terms of being signaling mutations) having these really divergent effects on the immunophenotype, was really interesting to us. And I think something that may end up explaining some of the results that we see in patients when they’re treated with cytotoxic therapy is that the FLT3 mutant AMLs are thought to be much harder to treat upfront whereas RAS-mutant AML is thought to be more responsive to initial therapy and this immunophenotype changes might be why.
Brittany: Do you think that studying these blood cancers opens up the door to understanding other types of cancer?
Linde: Absolutely. I think that’s been the one interesting thing coming from a solid tumor background into the blood cancer field and maybe eventually making my way back into some solid tumor space. There’s a lot that can be translated from solid tumor to liquid tumor and liquid tumor to solid tumor. And I think that by doing single-cell experiments like this, we know that solid tumors can be very heterogeneous. And there are multiple mutations, sometimes a lot more mutations than what we find in leukemia patients. Being able to really understand what mutations are working together, and what mutations are really co-occurring in on a single-cell level, I think is going to be really important.
Linde: Currently a lot of the solid tumor models are taking the common driver mutations or very frequently mutated genes and putting them together, and asking what those do in a cell. But I think being able to really know whether those two happen together in a patient is going to be really important and more accurately model their disease development.
Brittany: You’ve done a lot of great work on AML. What other projects still need to be done to better understand this disease and positively impact patient care?
Linde: There’s been some work, particularly from some groups out at UCSF and Stanford, where they were looking at patients that had undergone therapy. And I think a huge question is how patients are responding to therapy and how their leukemia is responding. Again, bulk sequencing is helpful but it doesn’t really give a clear picture of what resistance mechanisms are brewing at this very low level that then take off and become the dominant clone that is involved in a patient’s relapse. We touched on it in our publication, but there have been groups that have really been focusing on that.
I think single-cell analysis becoming part of the monitoring structure of clinical trials is going to be important in learning what those resistance mechanisms are moving forward so that they are caught at a very early stage and hopefully prevented from expanding to a point where the patient’s disease recurs. I think a similar situation is looking at the cases where patients have undergone remission or are in this minimal residual disease (MRD) situation where the disease is either detected at a very very low level or isn’t detected anymore. And being able to continue to monitor those patients for mutations that are slowly starting to creep up is important. I think bigger studies on the connections between the immunophenotype and genotype are going to be important. And we’re really excited to do a lot more of this work with the bigger antibody panel that Mission Bio has just released very recently.
I think that’s going to give a lot of resolution to our initial studies, but hopefully, we can continue to build on that. I think the other piece that is also really important is being able to connect DNA and the immunophenotype with what’s going on at the gene expression level. And so I think adding different multi-omics to the DNA plus protein platform is something we are super interested and excited about.
“Seeing how people continue to use the Tapestri Platform to build on the ability to ask really exciting questions — like when a cell picks up a mutation, what’s going on? I think that’s what this platform allows us to do. And I think it’s a really exciting time to be involved in it.”
Interested in hearing more about Dr. Linde Miles’ research? Watch this on-demand webinar.