Why Does Treatment Fail? by W. Gillies McKenna, MD, PhD

SLIDES & TRANSCRIPTS
Wednesday, February 16, 2000

Why Does Treatment Fail?
W. Gillies McKenna, MD, PhD

Slide 1:

Dr. TEPPER: For the second talk today we have Gillies McKenna from the University of Pennsylvania.

Dr. MC KENNA: I would like to thank Joel for inviting me today. My talk is actually going to cover some things that are fairly similar to the things that Carmen has just covered, except in relation to radiation therapy. We are also trying to understand if there is a molecular basis for this.

Slide 2:

Before I start I would like to acknowledge some collaborators, particularly Eric Berhardt, Ruth Machone, Elizabeth Marlon in our group at Penn, who have worked in some of the studies I will show you and, also, in the last couple of years we have been working closely with the group at Merck Labs, Jay Gibbs, Nancy Cole, and Alan Olaf when he was there before he went to Dupont.

Slide 3:

Our work derives from this observation originally by Edmund Malais, that if you take cells from tumors and look at them in vitro, that is, in the absence of any physiological effects and you just look at their sensitivity to killing by radiation, what you find is that pretty much the ranking that you find in in vitro sensitivity corresponds very closely to what you observe in clinic in the patients in terms of your ability to treat the tumors, leading Malais to postulate that there were intrinsic genetic factors in the cell that were really determining their sensitivity to treatment.

Slide 4:

Some work has confirmed this in at least one setting. Kathy West in England looked at patients being treated for squamous cell carcinoma of the cervix where she determined their sensitivity to killing by radiation in vitro in specimens removed from the patients before they received any treatment. The patients were treated with radiation alone, no surgery, no chemotherapy and again she found a very striking correlation between their sensitivity to treatment as determined in vitro and the survival of the patients months after treatment was delivered.

Slide 5:

To try to look at this we looked initially to see if we could find any cloned genes that we could use to alter radiation sensitivity, and what we and many others found about 15 years ago was that if you transfected the ras oncogene into primary cells in tissue culture that you could pretty reliably induce a radioresistant F phenotype.

Slide 6:

The question was did this have any relevance to human tumors. If it had relevance it could be an important effect because other than p53 ras is of course the gene that is most frequently mutated in human cancer, and it is mutated at high levels and in some GI cancers, for example, pancreatic colorectal tumor you see very high frequencies of ras mutation.

Slide 7:

So it would be relevant for this audience. When we looked to see what the mechanism of this was in the in vitro model what we discovered, even though we think of DNA as being the target for radiation damage, there was, in fact, no difference that we could detect in the levels of radiation-induced damage or in the kinetics or extent of repair of the damage in cells that were expressing ras. While the damage may be induced at the level of DNA it is not that damage, per se, that is leading to the altered sensitivity.

We found a couple of things that did correlate. The cell cycle delays in the presence of ras were different, and there was, also, protection from radiation-induced apoptosis when ras was expressed.

Slide 8:

To see if this was relevant for human tumors, since it is hard to make these paired samples of human cells, we collaborated with Eric Stanbridge and took a series of human tumors which were known to express ras mutations and then specifically knocked out the mutant ras allele, and in this case we are knocking out only the mutant ras allele. The normal remaining allele of the other ras gene is still present, and the other members of the ras family are not manipulated in any way, and we did this in tumor DLB1 which expressed the K ras mutation.

If we knocked out K ras, we could show that the cell became more sensitive to radiation.

Slide 9:

Here is another human line HT1080 which expressed an N ras mutation. This is the cells derived from the tumor, quite radio resistant. If we knock out N ras, the cells become quite sensitive. If we take this N ras knock out and now put N ras back in, we can restore the resistance.

We thought that this was evidence that in human tumor systems we were seeing a similar effect.

Slide 10:

What we have seen is that this then presents a fairly attractive target to try to manipulate sensitivity not only because ras mutations are frequent in human tumors but also because in many human tumors ras activity can be up regulated even in the absence of ras mutations if there are upstream events of ras that are mutant or disregulated.

Slide 11:

To be able to target ras we are fortunate in that we now understand that there are two things that are necessary for ras to be active. One is for binding of GTP. This is of course, what happens in the mutation is that ras becomes permanently locked in this GTP bound state, but in addition ras has to be inserted into the cell membrane by a farnesylation by a lipid modification of the protein. There is nothing that you can really do about the GTP binding of mutant ras, but it turns out that you can target the ability of mutant ras to insert in the membrane,

Slide 12:

and we can target that because we now know that this lipid modification occurs by an enzyme, farnesyltransferase which recognizes the 4-amino acid sequence in the C terminus of ras and attaches a lipid onto a C terminal cysteine. After you get this lipid modification ras will then undergo proteolytic degradation of these three amino acids in carboxymethylation. However, these last two steps are not necessary for ras activation. It is only the lipid modification that is necessary for activation of ras and recently a number of investigators in drug companies have derived drugs that will inhibit more or less specifically this enzyme farnesyltransferase.

Slide 13:

We obtained from a number of sources some of these drugs. This is a drug made by Andy Hamilton at Yale, FTI277, and in our rat embryo system we could show that treatment of the cells with this drug would indeed radio sensitize them so that when we used the drug to down regulate expression of the mutant ras we could see radiosensitization of the cells in vitro.

Slide 14:

However, what we really wanted to do was to determine whether these drugs could be used as radio sensitizers in vivo and could they be used in fact as radio sensitizers of human tumors in vivo.

Slide 15:

To do that we used a xenograft model placing human tumors in nude mice that were either expressing or not expressing ras mutations. One of the studies that I will show you is a regrowth study. We did a number of studies to look at the sensitization, but I will show you the regrowth data, and this is basically the protocol.

The mice were treated with implantable pumps so that they were receiving drug continuously throughout the period of study. The drug treatment began 3 days before irradiation because we knew for human tumors the kinetics of turnover of ras are such that it takes about 24 hours to drive the mutant ras out of the cell membrane. We started drug treatment 3 days prior to irradiation, and drug treatment was continued for a week. It extended beyond the period of irradiation, and then we measured tumor volumes,

Slide 16:

and these are the data that we obtained.

This is T24. This is a human bladder carcinoma that expresses an H ras mutation. Tumor grows quite rapidly in nude mice and will kill the mice relatively quickly. If you treat with radiation alone, here at dose of 6 gray, a relatively modest dose of radiation you do see a delay in the growth of the tumor.

However, after somewhere between 2 and 3 weeks the tumors will take off again with essentially the same kinetics and kill the mice. With the drug alone, this is L744832, a drug that was made by Merck, you see a delay in the growth of the tumor with the drug alone.

There are two reasons for this. One is the drug is cytostatic, and the drug does have some cytotoxicity by itself, although it is limited cytotoxicity, and notice in these experiments we deliberately chose a dose of the drug that would give us a growth delay which was essentially identical to what we saw with the single dose of radiation.

When we combined the two treatments, FTI plus the drug, we now saw this effect a very much more substantial growth delay and in fact, we could cure a substantial cohort of the animals with this single dose of 6 gray, and we have done with Rosy Myck, a statistician at Penn, an extensive analysis of this and although you cannot tell from this data that it is truly a synergistic interaction we are pretty satisfied that it is indeed a synergistic interaction.

We regarded this as proof of principle that FTIs combined with radiation could show a radiosensitizing effect in cells that expressed ras mutations and unlike the data that Neal Rosen reported with FTIs alone in human tumors we have seen no evidence with radiation effects that you get an effect in cells that do not express ras mutations, and we have now looked, not exhaustively, but we have looked at 8 of 10 cell lines with ras mutations and a similar number without, and we have never seen an interaction in the cell that doesn't express ras nor have we ever failed to see an interaction in the cell that does express ras if we are capable of inhibiting ras fernicilation. Based on this we have actuallycompleted a Phase I clinical trial which I am not going to discuss because it will be reported at ASCO in the spring.

Slide 17:

Interestingly, the effect that we saw was rather greater than the effect that we expected to see based on our in vitro data, and this led us to ask if there were other effects that were occurring in vivo that we couldn't see in vitro and specifically we asked whether the FTI was having any effect on the micro-environment of the tumors because, as Joel said, we, as radiation oncologists, strongly believe that in addition to genetic effects there are physiological effects that occur in tumors that affect their response to treatment. So we looked at a couple of these areas. One is on oxygenation of the tumors, and one is on the vascularization.

Slide 18:

A similar model using a variety of lanes that express mutant ras, both human tumors and murine tumors and colon carcinoma that expressed wild-type ras

Slide 19:

and unfortunately I am not sure this shows very well, this is using EF5. EF5 is metabolized by cells in hypoxic environments and it then becomes bound to intracellular protein. The red that you see here is binding of EF5. This is not necrosis. The drug must be metabolized by viable cells. You see no binding in necrotic area. These are viable cells but which are hypoxic, and in the control cells you can see that there is extensive hypoxia in this human bladder carcinoma.

After 3 days of treatment with the FTI inhibitor this hypoxia disappears,

Slide 20:

and you see the same effect with this prostate cancer. The tumor is extensively hypoxic in the control animals and here after 7 days of treatment, but we saw it also after 3 days, the hypoxia essentially disappears.

Slide 21:

In contrast, however, when we looked at this tumor, colon carcinoma that expresses wild-type ras in the controls, as you can see the tumor is also extensively hypoxic, but in this case treatment with the drug has absolutely no effect on the hypoxia within the tumors.

Slide 22:

This is this data quantified. We can use fluorescence intensity of VF5 as a measure of hypoxia. Here in the bladder carcinoma you saw extensive hypoxia in the control tumors and after treatment with the drug this hypoxia essentially disappears. Each bar here is an individual tumor and the P value for this is .0024.

If we look in HT29, a colon carcinoma that expresses wild-type ras, there is absolutely no difference between the control and the treated tumors. FTIs appear to have a specific effect on the physiology of these tumors.

Slide 23:

The question is can we make any sense of this in terms of the signal transduction pathway that ras is involved in? This unfortunately is where it becomes complex because ras is an early member of signal transduction pathways and talks to multiple downstream pathways and we also know that there is cross talk within these pathways.

Slide 24:

I am showing you this not that you can read it because I know that you can't, but even to single out ras is a vast oversimplification of what is going on even in the average tumor. This is from a recent and I think quite brilliant paper by Bob Weinberg in the millennial issue of Cell looking at what we know about what he calls the integrated circuit of the cancer cell at this point in time.

Slide 25:

However, we have tried to do it, and there are a couple of approaches that you can take. One approach which I will show you is you can use now, we have some fairly specific inhibitors of various members of downstream signal transduction members of ras, and so we can say if we inhibit you know a downstream member of a pathway would it have the same effect as inhibiting ras itself. Here we have used the drug PD98059 which is a fairly specific inhibitor of map kinase and, in data I am not going to show you, we can show that in fact map kinase is completely inhibited by this concentration of drug, but as you can see inhibiting map kinase has absolutely no effect on the sensitivity of these cells.

Slide 26:

In contrast, if we inhibit PI3 kinase, we now see an effect of PI3 kinase on the sensitivity of cells and in a dose-responsive manner, and we have done multiple experiments like this. We have also done similar experiments using dominant and dominant-negative members of downstream pathways. We have used Michael White's effector loop domain mutants of ras which are capable of interacting with some pathways but not others, and the data are mostly consistent.

Slide 27:

I want to try to summarize it in my last slide, and the summary still contains some ambiguities.

I mean I have shown you that ras appears to be able to modify the sensitivity of cells, and we have data with the FTIs that strongly supports that and we have now pretty good evidence that upstream signal transduction members may also be signaling through ras to alter radiosensitivity using inhibitors of EGFR kinase, for example, or other data that has looked in leukemic and lymphoma cells on inhibiting the abl kinase. Upstream the data are relatively consistent. However, when you try to go downstream the data are not completely clear at this point in time.

Tony Drischillois group at Georgetown has suggested that raf may be important in this downstream effect because they have data using antisense to raf that they can alter cellular sensitivity to radiation.

However, in my own lab when we have tried to inhibit signal transduction members downstream of raf, we have failed to confirm this result.

None of the downstream inhibitors that we have looked at is capable of altering radiosensitivity. However, what I did show you is that we had data that inhibiting PI3 kinase could give you the same effect that we saw by inhibiting ras which is attractive in that we know as Carmen talked about that this is a cell survival pathway.

We know that going beyond PI3 kinase to P70S6 kinase doesn't appear to be the pathway because rafomycin(?), which will inhibit this, doesn't affect cellular sensitivity to radiation.

Daphne Hass Cogan at UCSF has some data that P10 may be altering cellular radiosensitivity via its effect on AKTPKB which would be consistent with our model, but what it is downstream we are still not completely certain.

Carmen talked about NF kappa B, you know as a possible downstream member of this pathway. It is, also, possible that it is feeding into the bcl-2 caspase loop in affecting apoptosis. I think this is clearly one of the critical next steps that we need to take to try to understand exactly what is occurring down here. I am pretty hopeful that we are making early steps in understanding what the critical molecular determinants of sensitivity to radiation might be and this may lead us to develop new clinical strategies. As I said, the FTI trial has already completed the Phase I trial, and we are in the process of designing the Phase II trial.

I suspect, as in chemotherapy, that we are not going to find a single target that we will be able to use, and the importance of trying to dissect this out is because I suspect that in practice we are going to have to try and inhibit multiple members of these pathways to get a really profound effect because none of the drugs are really going to be able to perfectly inhibit the pathway that you are trying to target.

(Applause.)