SLIDES & TRANSCRIPTS
Monday, May 12, 2003

The New Genetics of T-Cell ALL: A Fish Tale

A. Thomas Look, M.D.

So, I would like to start with a little overview of work from Adolfo Ferrando in my lab, that is published in the article shown on the bottom of the slide.

We have divided now T cell ALL into five different molecular pathogenetic groups. Each of these pathways is a multi-step pathway, incompletely defined at the present.

As you can see, one prominent feature is activation of an intact oncogene, at least one, and in some cases two, often as you know, by chromosomal translocation.

Richard Bayer first, and then Adolfo, has shown that many of these genes can also be activated by upstream mechanisms independent of chromosomal translocation.

As I said, the main feature of these proteins, like HOX-11 or TAL-1/SCL are activated as intact proteins. So, it is clearly their mis-expression in T cell lineage lymphoblasts that contributes to transformation.

The last group, you can see here, the MLL-ENL group, a rare subset of the MLL group that you just heard about from Cheryl Willman, is clearly quite different.

Of course, in this case, you would have to have the chimeric fusion protein from the 11q23 translocation. Here certain HOX genes -- Myc-1 and others -- are activated, and not the genes in the predominant top four groups.

So, you can see a common feature of the first three groups is inactivation of INC4a and ARF, often through homozygous deletion on 9p, and I will call your attention to Myc.

So, you can see, at least in the first three groups, it looks like Myc is very central. It is upregulated in each of these groups.

In some cases, there is a translocation into the Myc gene, together with other mechanisms of activating, for example, TAL-1 and LMO2.

Myc-N seems to be upregulated specifically in the LYL1 group, a BHLH protein related to TAL1/SCL, and you can see in this case other tumor suppressors, at yet undefined, seem to be involved.

So, let's focus on Myc then. We decided if we wanted to model T cell ALL in man, a good place to start would be a transgenic over-expressing Myc, with the hope that we would then acquire other types of mutations as the leukemia developed.

TOP

So, we chose the zebra fish as a model. I just want to briefly summarize some of the advantages of this model.

A mating pair can have very many offspring per week, which is an enormous advantage for genetic and high throughput drug screen-type assays.

They are transparent, as you will see in a minute, a nice feature for developing a transgenic model. Transgenic technology is developed, they do certainly get cancer.

The blood developmental program is highly conserved. Much of this has come from work in Lynn Zon's laboratory at Children's Hospital.

Finally, the two main advantages, that I will come back to at the end of my talk, the zebra fish system is amenable to forward genetic analysis, like you have read about for years in Drosophila and C. elegans.

So, we think that this feature in a vertebrate might be quite useful for defining downstream molecular pathways.

Mark Fishman and Stuart Schreiber have shown that these animals are amenable to high throughput drug screens that can be arrayed out in plates, and small molecules can be screened directly to identify active compounds.

TOP

So, what about the model? As you see, this was recently published. So, you are certainly welcome to go and look at this in detail. It sort of put all the information in that paper in one slot.

So, what happens is, with a fusion transgene, in which the mouse Myc gene is driven from a zebra fish tag 2 gene promoter that we obtain from Shuo Lin at UCLA, and there is a fusion with GFP, so that the tumor cells will light up with GFP and fluoresce under a fluorescence microscope.

So, Shuo Lin had already found this promoter as highly specific for T cells, presumably, B cells also, although they are quite hard to identify in the fish.

So, when one injects this into a fish, David Langenau, a graduate student in the lab, was able to obtain a stable transgenic line in which the thymus is visible from five days of life, as two green dots on each side of the fish. The thymus is very lateral in the zebra fish throughout life. It doesn't merge in the midline.

You can see that, by 22 days, T cell lymphoma is clearly developed, so that there is spreading out enlargement of the thymus and spreading outside. This progresses rapidly over the coming weeks.

By 60 to 120 days, the fish will succumb of massive leukemia involving enormous spread throughout the fish, and the muscles around the head and neck, and replacing the kidney marrow. The bone marrow is actually in the kidney in the zebra fish.

TOP

So, this is published. I wanted to show you a couple of unpublished findings that have us pretty excited.

I didn't show you at the beginning, but the four pathways, or really five pathways, are broken out in a pie diagram here.

So, you can see that the most common type of T cell ALL is the group that activates TAL-1/SCL along with either LMO1 or LMO2, often with INK4A deletions.

We haven't been yet to define the molecular signature of the high risk patients, but most of them reside in this group. So, this is a mixture of better risk patients and the high risk group.

Obviously, this is a group we would really like to model using our zebra fish model.

TOP

So, David Langenau took a look at a series of tumors that had arisen in the fish, and you can imagine that we are quite delighted that SCL TAL-1 and LMO2 seem to be activated in each of the clonal T cell leukemias tested so far.

It looks like, by over-expressing the myc gene, we know these leukemias are clonal, with clonal rearrangements of the T cell receptor alpha locus, and now we know that they mis-express both SCL and LMO2.

We don't know if they actually have a chromosomal translocation, or if this aberrant expression is mediated through upstream mechanisms. We certainly think other mutations must be involved, since the leukemias are clonal.

TOP

This shows the same thing by in situ hybridization with RNA probes, showing the leukemia cells in the kidney -- these are the renal tubules, instead of the bone marrow spicules -- showing, with the antisense probe, high levels of expression of these cells compared to the sense probe for both SCL and LMO2.

TOP

So, what do we hope to do with this model? As I mentioned in the beginning, one potential advantage of working in the zebra fish system is their very small size and the reproducibility with which they develop the leukemia.

So, what we are trying to develop is a high throughput assay where you can put, say, six to ten transgenic fish as, let's say, about two to three weeks of age -- we are still working out the exact time -- at a time when they are less than a quarter of an inch long, into wells, array them out and then screen small molecules directly.

This has already been done, as I said, by Mark Fishman and Stuart Schreiber to mimic development defects that, of course, have been studied for years in the zebra fish. So, this is the kind of thing we are looking for, regression versus the usual progression.

TOP

A second advantage that I alluded to in my second slide is the idea of modifier screens. So, for this, I would like to illustrate this idea, rather than showing you a complex genetic screen diagram, with a simple Kaplan-Mayer curve.

So, here you see the fish, the transgenic fish with the Myc transgene, and this is actually now, we are thinking not survival, but the onset of T cell lymphoma.

So, let's say this is two weeks to three weeks, in the unmodified fish. So, what we can do is a genetic screen, mutagenizing the fish, and then observing clutches of fish from mutagenized parents, looking for enhancer mutations that will accelerate the onset of T cell leukemia.

So, the terminology gets a little bit confused here. Actually, enhancer mutations, we think would be either tumor suppressor genes or genes that increase the overall rate of genetic instability.

So, that might be nice, to discover a previously undiscovered tumor suppressor, of which we know there are at least 10, based on LOH studies, that remain to be found for T cell leukemia in man.

Then, of course, the potentially even more interesting idea, which I think the zebra fish is probably the only model that you could try to conduct such screens, would be screens to look for suppressor mutations.

So, these would be ones in which the fish did not develop leukemia nearly as rapidly as in the wild type, but either didn't develop it at all or had markedly delayed development.

These would be, then, affected by suppressor mutations, inactivating genes and proteins downstream of Myc somewhere, that would paradoxically prevent the tumor from forming, but hopefully not affect the health of the fish overall.

So, these, obviously, would be potential proteins from which to try to develop drugs that would inhibit them through small molecules, mirroring the effect of the genetic mutations.

TOP

So, I will stop there, and just mention all the folks who contributed this work. Primarily David Langenau somehow didn't even get on this version of this slide, with help, as I said, from Adolfo Ferrando and many others in my lab, Ed Prochownik, who is head of the myc GFP fusion clone, Nickolaus Trede, Lynn Zon and David Traver in Lynn Zon's lab has been enormously helpful throughout this project, Shuo Lin, who sent us the rag2 promoter, our pathology colleagues at Brigham and Women's who did all the hematopathology, and particularly Max Loda, who helped out with the in situ hybridization that I showed you. So, I will stop there, and I think we have time for questions.

TOP