Whole Genome Doubling (Tetraploidy) in CLL and Richer’s Transformation

Transcript:

Dr. Jennifer Woyach: Hi, everybody. My name is Jennifer Woyach, from the Ohio State University, and I’m excited to talk to you today about our research looking at whole genome doubling, or tetraploidy, in chronic lymphocytic leukemia and Richter’s transformation.

Richter’s transformation is a transformation of CLL to a more aggressive lymphoma. And although we think about this transformation as being relatively sudden, it’s more of a spectrum where the CLL cells start to take on characteristics that make them behave less like typical CLL, and more like an aggressive lymphoma, with more lymph node involvement, more whole-body or constitutional symptoms. And this is associated with a number of genetic abnormalities, which you can see here. And overall, they, in general, make the cells more genetically unstable.

About 15 percent of Richter’s transformation is associated with a specific gene alteration that’s called tetraploidy, which is where all of the chromosomes get doubled. This is often associated with other high-risk gene mutations like TP53 mutations. And we found a number of years ago that tetraploidy in CLL cells, so if the CLL cells show doubling of the chromosomes, that’s associated with a high risk of Richter’s transformation in patients who are undergoing treatment with ibrutinib (Imbruvica).

Tetraploidy is something that occurs in many types of cancers, both in blood cancers, as well as solid tumors, and it makes the cells more easily able to acquire additional gene abnormalities like mutations or other chromosome changes. And the goal of our research is to understand the basis and the consequences caused by tetraploidy in CLL, and how this can cause or help facilitate transformation to Richter’s. Our hypothesis is that the tetraploid clone that we can find in CLL is actually a precursor to Richter’s transformation, and that it has unique genetic and other features that we typically associate with Richter’s.

This is some work that was done by Anthony Mansour, who was a resident with us at Ohio State, and is currently a heme/onc Fellow at City of Hope. We looked at about 80 patients that were treated at the Ohio State University, who were shown to have tetraploidy on chromosome analysis. And we found that over time, actually, about half of them developed Richter’s transformation. And that the patients who developed Richter’s transformation, it tended to be associated more commonly with other mutations like TP53 mutations, deletions of a gene called CDKN2A or N2B, which are also associated with Richter’s.

In order to study tetraploid in the laboratory, we made cell lines tetraploid by treating them with chemicals that interrupt the cell cycle, and we used two well-characterized CLL cell lines called OSU-CLL and MEK-1. And then, we separate out the tetraploid cells from what are called diploid cells, which are normal copies of the chromosomes. And all the lab work I’m going to show you has been done by Samon Benrashid, who is a graduate student who works with me.

These are called Circos plots, and they’re used to show changes in the genome. And we did a procedure here that’s called optical genome mapping, where we unwind the DNA of cells, and then fluorescent probes are attached that can tell whether specific parts of the DNA are normal or abnormal. And then, a special microscope is used to capture those fluorescent probes, to tell where and what type of changes have occurred. So, all the numbers that are on these circles correspond to different chromosomes, and each color within them is a different type of genetic abnormality.

Here, we’re comparing a cell line OSU-CLL that is normal wild type, normal number of chromosomes, to ones that we made tetraploid. And you can see pretty clearly that the cells that are tetraploid start off with more genetic changes, in addition to just having the doubling of the chromosomes. Then, we took the cell lines and grew them in culture until they divided 20 times, and looked at the genome again, and we saw that the tetraploid cells acquire additional abnormalities more quickly than those that have normal amounts of chromosomes.

We then looked at cells from CLL patients with tetraploid, where we collected samples over time, and we separated out the tetraploid cells and did this optical genome analysis again. And you can see, compared to the sample from 2008, a couple years later, you can see a lot more genetic changes.

To understand these specific genetic changes that were occurring, we took samples from patients with tetraploidy, where we had samples collected over time. We separated out the tetraploid cells from the normal diploid cells, and then we performed RNA sequencing of each of those different compartments at the different time points. And this is just a representative sample from a patient where we had cells stored in 2011, when tetraploidy was first noted, and 2013, when the patient developed Richter’s transformation.

When we look at specifically the early time point, here, we’re looking at the normal diploid cells compared to the tetraploid cells, and we see that there’s actually a number of differences between the normal chromosome cells and those with double chromosomes. We see a lot of expression of genes involved in inflammatory responses that we often associate with Richter’s transformation.

Now, we’re looking at our later time point. And we, again, are looking at diploid, or normal chromosomes, versus tetraploid. And we can see that the cells look even more different at this late time point. Over here are some pathways that are different between the diploid and tetraploid cells, and the red asterisks are denoting pathways that are commonly associated with Richter’s transformation, that also are seen in our tetraploid cells.

And then, finally, when we look over time at just the tetraploid cells, we see evolution of the cells. So, genes become more upregulated over time, similar to what we have seen in the literature when we compare Richter’s transformation cells to CLL cells.

So, what we’re showing from this is that the tetraploid cells start out a little bit more – have gene associations that are typically seen with Richter’s. We see those get even more pronounced over time. And so, one of the reasons to do this is to see whether there might be targets that could be used for therapy here. And one of the genes we see consistently overexpressed in our tetraploid cells is called PLK1. PLK1 is a gene that’s very important in the regulation of cell division. It’s actually dysregulated in a lot of cancers and is very tied to activity of TP53.

In addition to our primary CLL cells, our tetraploid cell lines also have higher expression of PLK1, which makes them a good mechanism to study this as a target. There’s a number of PLK1 inhibitors in clinical development, although none have yet been tested in CLL.

Here’s some data showing you that when we treat our cell lines, so our OSU-CLL cells that have normal chromosomes – that’s the 2N, or tetraploid cells, 4N. And you can also see there are OSU-CLL cells that say p53 knockout. Those are actually also tetraploid cells that became tetraploid when we knocked out TP53. When we treat these OSU-CLL cells, or MEK1 cells, with a PLP1 inhibitor, we see that all the cells really have some sensitivity to the drug. But those that are tetraploid, which are in red and in green, are increasingly sensitive to treatment.

And interestingly, when we treat our CLL cell lines with a PLK1 inhibitor, we can actually reverse some of the genetic changes that we see tetraploid cells acquiring over time, suggesting that we are making them act more like cells with normal chromosomes.

What we found so far is, tetraploidy is a major risk factor for Richter’s transformation. That tetraploid cells develop gene abnormalities at a faster rate than those with normal chromosomes. In primary CLL cells, tetraploid cells are different than their diploid counterparts. They have gene expression that we associate with Richter’s transformation, and they evolve over time. And PLK1 is a target that’s overexpressed in tetraploid cells, and we think this might be an opportunity for intervention.

This is a project that is obviously still underway. And we’re hoping that by understanding more and more about tetraploidy in both CLL and Richter’s transformation, we can develop better therapies to treat Richter’s, or potentially even prevent it from occurring. Thank you very much.