Resistance to Therapy: Cell Cycle Regulation and Growth Factor Signaling as New Therapeutic Targets in Gastric Cancer by Gary K Schwartz, MD

SLIDES & TRANSCRIPTS
Tuesday, March 6

Resistance to Therapy: Cell Cycle Regulation and Growth Factor Signaling as New Therapeutic Targets in Gastric Cancer
Gary K. Schwartz, MD

Slide 1: Introduction

This morning I will talk about a topic that really, I think, is pertinent to the development of new therapeutics in gastric cancer. I think in the last year we have seen major developments in gastric cancer therapy, especially in the adjuvant setting. David and Peter and others have alluded to the fact that, despite advances in adjuvant therapy, our patients still die of metastatic disease.
I think this, in large part, reflects the failure of chemotherapy, despite some advances in the last several years. The question is, how does chemotherapy really work, why does it fail, and how can we make it work better. The way we are going to approach this is by looking at the cell cycle and growth factors, and seeing how we can translate this area of development into clinical cancer therapy.

Slide 2: Chemotherapy and Radiation

So, what happens with chemotherapy? Well, this was shown in some part yesterday. I am going to go through again briefly. Chemotherapy and radiation are supposed to damage the cell and damage the DNA in one paradigm, for the induction of a process we call apoptosis, which is a process of programmed cell death. We think we should all agree that chemotherapy kills cancer cells through this mechanism of different cellular events.

Slide 3: p53 Induced

The DNA gets damaged, p53 gets induced.

Slide 4: p53 Releases Bax

P53 released BAX, a pro-apoptotic factor.

Slide 5: Bax Displaces BXL-2

BAX then displaces BCL-2, an anti-apoptotic factor from APAF-1.

Slide 6: APAF 1 is Released

APAF-1 is then released into the cytoplasm. BAX itself enters the mitochondria. It then forms pores in the mitochondria, resulting in the release of a factor called cytochrome C. Cytochrome C then sees APAF-1.

Slide 7: CYTO C

APAF-1 is now in the cytoplasm as well.

Slide 8: Merge with Caspase 9

APAF-1 and cytochrome-C now merge together and also come together with another caspace called caspace-9.
Now, these caspaces are cysteine proteases, which are the final common executioners of the cell death of gastric cancer and other tumor types.

Slide 9: Complex Forms

A complex is formed with cytochrome-C, caspace-9-APAF-1.

Slide 10: Inactive Procaspase 3

It then sees another caspace called pro-caspace, a pro-enzyme inactive. It then cleaves pro-caspace-3 into the active form caspace-3.

Slide 11: PARP

Caspace-3, through unknown mechanisms, enters the nucleus of the cell, sees a protein called PARP -- poly-ADP ribose polymerase.

Slide 12: Cleaves PARP

It cleaves PARP, a marker of apoptosis, and I will show you this in gastric cancer. It also leads to fragmentation of the DNA in the nucleus, resulting in chromatin condensation and morphologically, under the microscope, you see these cells become a fluorescent blue pattern. I will show you some slides regarding this with chemotherapy, or inability to do this, with standard chemotherapy drugs. This whole process is called apoptosis. We are going to focus on caspace-3 PARP cleavage, this particular phase of the apoptotic cascade. There are many ways to examine this, but this is our focus for this morning.

Slide 13: Cell Cycle

Now, how do we induce apoptosis? How can we modulate this process to further advance the treatment of gastric cancer?
Well, our approach has been focusing on the cell cycle and cell cycle regulation. In the cell cycle, there are proteins or kinases called cyclin-dependent kinases or CDKs.
Now, these CDKs are responsible for the movement of the cell through the cell cycle. An activation, for example, of CDK-4 or CDK-6 will move the cell from G-1 to S. Activation of CDC-2 will move it from G-2 to M. Without these CDKs, the cells cannot move, the cells cannot live. An implication of inhibiting the CDKs is induced apoptosis.
Now, the CDKs themselves are activated by proteins called cyclins.

Slide 14: Cyclin Dependent Kinase Inhibition and Apoptosis

You can think there may be different ways of turning off this process.
If you turn off this process, you induce apoptosis. So, you would target the cyclins. This could be done by different ways. There is no activator of the CDKs. The cells cannot move into the next phase of the cell cycle, and the cell undergoes growth arrest.
It has been shown, predominantly in leukemic cells, and in some solid tumors, that this process will induce apoptosis. You can knock this out with an anti-sense construct, different mechanisms for enhancing the degradation by ubiquitin produced on cascade. This has been developed in different tumor types.

Slide 15: Intrinsic Factors

Another way to take this forward is using intrinsic factors. Not only are they activators of the CDKs, they are intrinsic inhibitors of the CDKs. Now, these inhibitors exist in the tumor cell. They bind to the CDK -- in this case CDK-2 such as p21 -- and they turn it off resulting, again, in growth arrest and apoptosis.
You could think, this could be modulated. You could transfect p21 into the cell, with this gastric cancer or colon cancer, induce a G-1 block, and the cells should die. We have taken a different approach.

Slide 16: CDK Inhibitors

Our approach is the development of small molecules. I am going to talk about three of these today. A lot of this will be about flavoperidol, which is in clinical trials. We will also talk a little bit about bryostatin-1, FL UCN-01. These are drugs that are being developed in my laboratory and others, and our target has been gastric cancer.

Slide 17: Flavopiridol

This is flavoperidol. It was actually first synthesized from a plant indigenous to India. It is now chemically produced in the laboratory. It is supplied to us by the NCI, and also given to NCI by Aventis Pharmaceuticals.

Slide 18: Inhibition of CDKs

Flavoperidol, as a drug, is a CDK inhibitor. It blocks CDK-2 and CDK-4 and 6. So, much like p21 or p27, we don't need those proteins to inhibit it. We can use this drug. The cells rest in G-1.

Slide 19: Inhibition of CDKs

Another target of flavoperidol is to inhibition of CDC-2, also called CDK-1, and results in G-2 block and arrest of the cells in G-2. So, this drug is not specific for any one CDK, multiple CDK sites, and substitutes for p21, p27 as a CDK inhibitor, but as a small molecule, not a protein.

Slide 20: Results of Phase II Study

Now, we were interested in flavoperidol for several years. We have actually done a study of this drug as a single agent in gastric cancer. This will be published in the April issue of JCO. It is a negative study.
At a dose of 50 milligrams per meter squared per day, given as a continuous infusion for three days every two weeks -- this was from the phase I design of Sendarowitz at the NCI. When this was given every two weeks, it was an inactive single agent in metastatic gastric cancer. We saw no response in 14 patients. Bad news. We saw unanticipated and unacceptable toxicity, including diarrhea, fatigue and phlebitis.

However, we did achieve in this study -- this is the good news -- a mean peak level of flavoperidol or 385 nanomolar, and that is sufficient to inhibit the CDKs. So, why did this fail? In gastric cancer, prostate cancer, lung cancer, colon cancer, every disease it has been treated in, this has failed. There has to be some reason. We are getting clinically achievable doses. As we were developing the clinical program with this, we directed a laboratory effort based on a hypothesis we generated in the laboratory in the early 1990s.

Slide 21: Inhibition of Colony Formation

Before I get to that, I want to show you one more slide. Why did it fail? This is actually studies from Bible and Kaufman looking at a series of cell lines with flavoperidol from the Mayo Clinic group, looking at single agent activity of flavoperidol in a number of tumor cell lines -- colon, lung, brain, here is a breast and here is a leukemic cell line. So, flavoperidol, in increasing concentrations, will inhibit colony formation of the cell. This should work, therefore, in patients. I mean, this is a fairly predictive assay.

Slide 22: Inhibition of Colony Formation

However, here is the problem. The dose of flavoperidol that we achieved clinically is 300 nanomolars. Here is 300 nanomolar on this growth curve.
If you then follow this up, you see we are very effective in killing colonies of leukemic cells, and that is where all the apoptotic models were done with this drug, in leukemia.
You follow this up and you see that many of these cells are still growing. They have still got colonies. They are not dead. In fact, if you remove the drug, the drug will grow. They are growth arrested, but they are not dead cells.

Slide 23: Hypothesis for Enhancement

The hypothesis was simple. Growth arrest by CDK inhibitors does not result in cell death. However, we hypothesized that if you could inhibit growth arrest with a CDK inhibitor and add a chemotherapeutic drug for radiotherapy, you should result in irreparable DNA damage and profound apoptosis. So, how are we going to study this in gastric cancer? When we entered the field, there were no human gastric cancer cell lines for American patients. There are Japanese cell lines, and we heard about TMK yesterday, but the first obstacle was testing it in American gastric cancer patients. It seemed like an easy thing initially, but they didn't exist.

Slide 24: Establishment of Cell Lines

So, we established our own, and these are the five cell lines we use in our laboratory. These are SK for Sloan-Kettering gastric tumor one, two, in order in which they were discovered and established in patients.
You notice proximal stomach, one of my favorites, GE junction. All of these have p53 mutations. This one actually had p53 null. This is the electromicroscopy of these cells. They have been fully characterized, we have published this data. We have looked at this p53 RB, proteolytic factors. I could go on and on. These are the five cell lines that became the basis of our laboratory program in gastric cancer and new drug development.

Slide 25: Mitomycin C Induced Apoptosis

We wanted to test the hypothesis. Would a CDK inhibitor with the conventional chemotherapy induce apoptosis where neither drug would do it by itself.
This is a hook stain. We looked for apoptotic cells. This is SK GT5. Here is the control panel. Flavoperidol, three nanomolars. No apoptosis. These are growth arrests. See, there are fewer cells, but they are not dead. They are still alive. This is why it fails clinically. Mitomycin, which was our first drug five or six years ago, not as interesting today, occasional apoptotic cells. Here is one, here are two, 10 percent.
You take the same dose of flavoperidol, the same dose of mitomycin, give them together and you get massive apoptosis. Up to 80 percent of the cells are now dead, not just arrested. These are dead cells. These will not grow. Here they all are. This has been published in Clinical Cancer Research several years ago.

Slide 26: Paclitaxel (T) Induced Apoptosis

Mitomycin is interesting. We are now developing Taxol, one of the newer drugs that Peter was mentioning in gastric cancer. I mentioned once before that PARP cleavage is a hallmark of apoptosis. We treated these cells. Actually, this is another cancer cell line, MK-N74, one of the Japanese lines. Nevertheless, taxol, at 100 nanomolars, does not induce PARP cleavage. We looked 24 hours later, maybe there is delayed apoptosis. You get a little PARP cleavage. However, with taxol, followed by flavoperidol, there is profound cleavage of PARP. You lose the 116-KD band completely and get a 90-KD band significantly. You get significant apoptosis with taxol followed by flavoperidol. We looked at some of the caspaces.

This is just some of the early data. Caspace-3, which exists in an inactive pro-enzyme, gets activated by forming a 17-KD band. You can see that with taxol followed by flavoperidol, there is about a two to three-fold increase in caspace-3, but other caspaces are even more activated than caspace-3. This indicated that if you take a cell that is resistant to apoptosis by chemotherapy, and add flavoperidol, you can open up the caspaces that are inactivated, drive them forward, turn them on, and elicit apoptosis in a cell that is resistant to the induction of apoptosis by chemotherapy alone.

Slide 27: Morphology

Here is the morphology. Here is taxol alone, here is taxol followed by flavoperidol. There is massive apoptosis. I don't think anybody would argue that this is not. We then treated these cells with ZVAC, a caspace inhibitor. You block the caspaces, theoretically, you shut off apoptosis and block the process completely. We did that. ZVAC, here, and the treatment with taxol flavoperidol, completely blocks the product, indicating again that flavoperidol turns on the apoptotic cascade, activates the caspaces in the presence of chemotherapy. You need the chemotherapy there, because flavoperidol will not do it by itself. You will get apoptosis or block it if you have a caspace blocker. This is just showing that, by taxol by itself, you also prevent apoptosis as a single agent with ZVAC.

Slide 28: Sequence Dependent

Now, one thing we learned, this is very sequence specific. If you give taxol by itself in gastric cancer cells, you get minimal apoptosis, flavoperidol minimal apoptosis, although growth arrest. Here is taxol with flavoperidol. Not much. The only way this works is sequentially. Taxol must precede flavoperidol to induce apoptosis. We counted the cells under the microscope. It is almost 60 percent. The reverse process, again, no apoptosis. Why is this?

Slide 29: Activation of cdc2 Kinase

One of the things taxol does, it activates a kinase called CDC-2 kinase. This is actually gastric cancer cells again, showing you that taxol activates the kinase, and this kinase is responsible for the movement of cells from G-2 to M. Flavoperidol inhibits the kinase as you would expect. Now, why is this important in the development of these drugs for gastric cancer therapy?

Slide 30: Cell Cycle Interactions

Taxol activates cyclin-B CDC-2, cells move from G to M and arrest in M. If this kinase is not activated, the cells don't go to G-2 and taxol can't even work as a single agent.

Slide 31: Cell Cycle Interactions

So, flavoperidol comes in, turns off CDC-2 cyclin-B, as shown here,

Slide 32: Cell cycle Interactions

and in the presence of taxol, which needs its activated kinase to move forward, taxol becomes ineffective. So, if we give these drugs too soon, since each drug is cell cycle specific, taxol has its cell cycle effects, flavoperidol has its cell cycle effects. If you give them the wrong way, you will actually antagonize the chemotherapy and block the effect completely. This is an important observation, I think, and is important in clinical development.

Slide 33: Phase I Study Paclitaxel, Flavopiridol

We have actually completed a phase I study of taxol, day one, three-hour infusion followed by flavoperidol day two, taking advantage of sequence specificity. I am not going to have time to talk about this phase I trial.
We were able to get up to flavoperidol doses of 70 milligrams per meter squared. We actually escalated taxol of 175 per meter squared on day one. That will be the recommended phase II dose.

We did see activity in esophago-gastric cancer. This is a patient who had a GE junction esophageal tumor. He could not swallow. He had tumor occluding the lumen of the esophagus. That was the pinpoint lumen, which he was able to swallow some fluids. These are the nodes before therapy, significantly enlarged. After two cycles of therapy, the lumen was now open and the lymph nodes were now half the size, down over 50 percent. This was a clinical PR. He also went into a pathological PR with continual therapy. We biopsied him and could find no viable tumor in his esophageal lumen. This patient did not receive prior tax, so you could argue this is a taxol effect. He went over 13 months. He eventually did fail. We had a patient who went on 19 months before she eventually did fail. We have had patients on the study who failed taxol, went on to taxol-flavoperidol, have had PRs, and based on this, in fact, we are now doing a phase II study in esophago-gastric cancer for taxol-refractory disease. The next study has just opened at our cancer center, based on some encouraging preliminary data from the phase I study.

Now, I want to talk just a few minutes about not just taxol. We have taxotere data, which I am not going to share with you today. All the taxanes appear to be enhanced by this drug. What about other drugs? There is SN-38 in gastric cancer. We have talked about novel therapeutics. What about these drugs with flavoperidol.
Again, PARP cleavage, one of the hallmarks of apoptotic events, this is SN38 by itself in this lane. As you can see, you are in gastric cancer, SN38. At a dose of 160 nanomolars, which is a lot of drug, this is the active metabolite of CPT-11. It does not induce PARP cleavage. If you give SN followed by F, you almost lose completely the initial band, get dramatic induction of the PARP product. It is not as sequence dependent as taxol. You get some of it in reverse, some of it together, but the best is with sequential SN followed by F. F by itself, no apoptosis, a little bit here, but generally not. Again, there is a clear association with activation of the caspaces. Here is caspace-3. There is significant activation from the inactive 34-KD pro-enzyme to the active 17 KD and 14 KD bands, most significantly with SN followed by F, another example of the induction of the caspaces in situations where chemotherapy isn't capable of doing this by itself in gastric cancer.

We actually took this into colon cancer, because this is where the drug is most currently used. Here is a xenograft model, CPT-11 alone. Basically, we were unable to complete eradicate any tumor with CPT-11 alone. CPT-11 followed by flavoperidol on different schedules, here is four hours. If you hold off flavoperidol seven hours later, we had a complete response in this animal, pathologically confirmed. Actually, this has been submitted and hopefully we will see this in Clinical Cancer Research in the next couple of months. These are completely -- we used the word cure in our paper. There were some concerns about our exuberance about this, but this is what we did report. In about a third of these animals, you cannot find detectable tumor, but in every CPT-11-treated animal, although there were regressions, everyone had residual disease. I should say that, based on this study, we have now opened a CPT-11-flavoperidol sequential therapy, a phase I study in patients with advanced cancer, based on these very encouraging in vitro and in vivo data. It is open to gastric cancer patients.

We have had a preponderance of colon cancer patients on the study, and we are seeing some interesting clinical data. I don't have time to go into that today. Radiotherapy and gastric cancer, Chris Willett here, does it enhance radiotherapy? We have just talked about this being such a big new treatment in gastric cancer. This is a complicated slide. Here is radiotherapy on gastric cancer cells with minimal PARP cleavage. Here is radiotherapy and flavoperidol. There is significant PARB cleavage and, again, dramatic induction of caspace-3, all with combinations of RT-flavo. There is also gemcitabine flavo in gastric cancer, another story. This gemcitabine flavo combination, in fact, is being tested at Dana Farber by Jeffrey Shapiro in a phase I study based on this data as well.

Slide 34: Byrostatin-1

Bryostatin, I am not going to talk much about. This is a very complicated drug. It is a macrocytic galactone. It is actually from the sea. I don't know if you watched ABC news last week. It is a moss-like plant that activates and inhibits other targets such as PKC, inhibits CDK-2 as well, inducing P21, inhibiting CDC-2 kinase, also by suppression of cycline-B-1, as reported in CCR about a year ago.

Slide 35: Phase I Study Paclitaxel, Bryostatin-1

This has also gone into phase I trial, not into phase II in esophago-gastric cancer at our center. It is given weekly with sequential therapy, paclitaxel followed by brio. This has been published on why the sequence is important even for bryostatin.
Again, it is sequential dependent. The paclitaxel must precede bryostatin in order to induce the effect. This is a phase I design. We are actually able to get to 50 micrograms per meter squared of bryostatin. We have had to cut back the dose because of myalgias, which is the main side effect of this drug.

Slide 36: UCN-01

This is actually a patient who had an unknown primary, although it was believed to be from the upper GI tract.
This is the PET scan. These are the multiple bone metastases when he started therapy and, after two months of treatment, you can see these bone metastases by PET scan have essentially resolved. He is off therapy now being followed by serial PET scan with no evidence of disease. UCN-01 is a storasporin analog. It came from the broth of streptomycin species. Again, it was developed initially as a PCK inhibitor, predominantly in micromolar concentrations. We found out later, by Ed Salzberg's group at the NCI, that in fact it is a CDK inhibitor at nanomolar concentrations, and this is a drug that we can achieve clinically.

Slide 37:G1-S Transition

UCN-01 is interesting. Also, it is a cell cycle drug, and works at G1S. The G1S transition is regulated by a group of factors, and predominantly RB. RB gets phosphorylated by the CDK, CDK-4, 6 and 2, and again by the cyclines.
The phosphorylation releases RB from E2F, E2F binds the DNA and induces transcription of a series of factors that are critical for many of the chemotherapies such as TS for 5FU, RR -- ribonucleotide reductase -- for gemcitabine, TK and CHFR for methotrexate. Now, why is this important for UCN-01 therapy?

Slide 38: UCN-01 Inhibits

UCN-01 actually comes and inhibits the CDK-4 and 6 complex. It also results in hypophosphorylation of RB.
The bottom line of all these effects of UCN-01 is to induce the G-1 block and, therefore, you don't get transcription of a number of critical factors that are targets for chemotherapy, but also the factors responsible for chemotherapy resistance.

Slide 39: 5-Fluorouracil

Predominantly fluorouracil -- and I haven't mentioned this drug yet, but I am going to talk to you in the concluding four or five slides on fluorouracil, and I think we will hear more about this later this morning.
TS correlates inversely. There is much of the data from gastric cancer, from the University of California group, correlates inversely with survival, and 5 fluorouracil-based treatment response to gastric cancer. TS levels are increased after fluorouracil exposure.
There have been many attempts clinically to develop this with direct TS inhibitors -- leucovorin, the FDM complex and finally tomudex, which directly inhibits TS at its catalytic domain. Why am I telling you about this?

Slide 40: Suppression of TS Protein Expression

Well, here are fluorouracil effects on gastric cancer. We treated gastric cancer cells with 5 FU and you get dramatic induction within 24 hours of TS.
I think it is pretty significant. You can imagine this being your patient with fluorouracil and TS clearly associated with fluorouracil resistance. However, in the presence of UCN-01, you have completely suppressed the protein expression of TS. Here is the fluorouracil control, and this is just to here to show you the protein loading.

Slide 41: Dose Dependent Decrease

Why is this important? UCN-01 suppresses the transcription of TS at the RNA level. This is a northern blot.

Slide 42: Regulate UCN-01

This is also the fact that you can regulate UCN-01. We show this as actually a post-translational effect. LLOL is the ubiquitin proteosome inhibitor. In fact, it will restore E2F1, and also restore TS. The bottom line is this, that UCN-01 activates the proteosome ubiquitin cascade, leading to degradation of E2F1, and loss of T2S at the transcriptional level. So, now we have a way of affecting the transcription of TS, and we believe also enhancing the effect of fluorouracil. Now, is this true?

Slide 43: UCN-01 Enhances

Well, here is SK-GT5, our original GE junction adenocarcinoma cell line. Here is fluorouracil. Again, not an effect on apoptosis. UCN-01, an occasional apoptotic cell. Here is sequential 5FU UCN-01. There is significant apoptosis, 50 percent in our series, and UCN-01, followed by fluorouracil, a reverse combination, shows an occasional but not as much. It is about 30 percent. The sequence seemed to be favored here.

Slide 44: Phase I: UCN-01, Fluorouracil

Based on this, we actually have a phase I design, which is currently open to patient accrual, giving UCN-01 and fluorouracil. Fluorouracil is given weekly as a 24-hour infusion. UCN-01 is given every month because this drug has a very long half life. It is given as a fixed dose continuous infusion for 72 hours a month, one, and then repeated as a 36-hour infusion, the second one.

This is actually some of the early clinical data with a patient with colon cancer. This patient received XLA-platinum, CPT-11, 5FU leucovorin. He failed all conventional chemotherapy. He came to us in a salvage program. He went into the study using UCN-01 fluorouracil. This is the baseline CAT scan. I think it shows the power of these sorts of combinations. This is after two cycles of treatment. I think you might agree with me that it doesn't achieve a CR, but it is at least on its way to PR.

Slide 45: Process Apoptosis

I am not going to burden you any more, but this is a complicated process, apoptosis. You can get there any different number of ways.
I focus on caspace-3. There are TNF cascades and yesterday there was discussion of TRAIL -- tumor necrosis-related inhibitory factor ligand -- which may activate some of these cascades. It was mentioned yesterday that TRAIL is a promising new drug therapy. There is a problem with this, that TRAIL also seems to induce apoptosis in normal hepatocytes. This may be a problem in this particular drug-development program.
There is fas ligand for enzyme B. There are multiple caspaces, all of which the bottom line is the activation of a series of internal proteins leading to apoptosis.

Slide 46: Drug Targeting EGF Receptor

I am only going to spend one minute on this. Really, it was talked about yesterday and I don't think it is worth going through it again.
There are four drugs that target the EGF receptor that are being developed in different diseases, C225, receptin, OSSI-774, which has just been offered by the NCI for clinical development for gastro-esophageal cancer, and ZD-1839, which has been discussed extensively as another targeting drug for EGF receptor.

Slide 47: Targeting - Expression Varies

EGF receptor exists on the cell surface. One of the questions in gastric cancer is, what is the expression. If you believe these drugs are only as good as the target, you have to have the target there to inhibit it, to get an apoptotic event or some sort of cell cycle effect.
In gastric cancer, this varies anywhere from 10 to 30 percent in the series, but if you look in JCO four or five months ago and her2/NEU is now being reported to be expressed in 90 percent of the tumors in gastric cancer.
I think reflects a number of things. Is it the antibodies used in determining immunohistochemistry. There are objective biases in evaluating IC analysis. There are qualitative issues that need to be addressed as we target these for drug development with the drugs that are currently available.

Slide 48: Targeting - Cellular Proliferation

Again, a potential target, the activation of these receptors, are based on the phosphorylation and the tyrosine kinase activation leading to cellular proliferation in the presence of the growth factor ligand.

Slide 49: Binding Induces Growth Arrest

C225 receptin binds to these receptors, preventing their homodimerization. One of the effects of these drugs, in fact, is to induce growth arrest and lead to G-1 block. This gets back to another way of inducing G-1 arrest and may explain why these drugs by themselves are ineffective as single agents. Growth arrest I do not believe is sufficient to kill the cancer cells.

Slide 50: New Drug Therapies

Again, OSI-774 and ZD-1839 are new drug therapies in contrast to the antibody approach, which I showed you in the previous slide.
These drugs inhibit the kinase activity,

Slide 51: Growth Arrest Possible

turning off tyrosine kinases here. However, again, growth arrest is possible but not cell death. I think one of the advantages of these drugs, if you combine them with chemotherapy, you can get apoptosis, which I think supports our model that growth arrest by itself is insufficient to kill the cancer cell.

The growth arrest in the presence of a cytotoxic can kill the cancer cell, especially in gastric cancer. Now, none of this has really been tested in gastric cancer with these drugs. I think clearly this needs to be evaluated. There are models for colon cancer and breast cancer, but the gastric cancer has not been developed.

I do think that this does lead to potential therapeutic implications in new gastric cancer therapies, targeting EGF receptor. I would argue that it shouldn't be as a single agent. It should be in combination with a cytotoxic chemotherapy, radiotherapy, whatever cytotoxic is going to be the treatment de jour.

Slide 52: Conclusions

In conclusion, our understanding of the molecular base of gastric cancer resulted in the identification of factors that are critical for the induction of apoptosis.

CDK inhibitors can augment the chemotherapy-induced apoptosis in cells that are chemotherapy resistant.
The interaction of these drugs relative to chemotherapy depends on an understanding of cell cycle physiology.

Therapies directed against growth factor receptors represent new therapeutic agents, overcome chemotherapy-resistant gastric cancer, and the clinical applications of these novel therapies is underway.

I want to get back to Dr. Gunderson's comment. Initially he said it took him all this time in his career to see that adjuvant therapies work in gastric cancer.
I promise Dr. Gunderson, we are going to see these drugs used soon in gastric cancer therapy. We just need to seize the moment. The moment is here. We just now need to develop these for gastric cancer treatments. I think we have opportunities with the NCI and the pharmaceutical companies to do this type of approach in the treatment of this really terrible disease.

Slide 53: Thank you

I will end on that final philosophical point. Thank you.