I want to speak about a very exciting new target -- the proteasome -- that has been proven and validated as a clinical target, because the first prototypic drug, bortezomib (Velcade), was approved for multiple myeloma by the US Food and Drug Administration (FDA) in 2003. We'll speak about that drug and about why multiple myeloma is a disease that lends itself to this new target.

But first we'll discuss the proteasome and what it does. The proteasome is ubiquitous. It is considered a garbage disposal -- the proteasome is responsible for degrading and eliminating proteins that are no longer needed. It also maintains -- in a posttranslational way -- homeostasis, by eliminating various proteins involved in regulating cell cycle, survival, migration -- certainly in cancer metastasis. So this is an abundant protein; it probably regulates as many as 80% of all proteins within the cell.

To target the particular proteins that need degrading, those proteins must first be ubiquinated through a series of enzymes that create a polypeptide chain of ubiquitin molecules. This ubiquitin tag then sends the targeted protein to the proteasome for elimination.

This process is very specific, and as you'll see here, the target that is ultimately ubiquinated is usually shielded. When this protein is damaged or ready for elimination, however, a lysine generally is revealed and can be targeted by adding these ubiquitin molecules.

There is a series of enzymes -- 3 families in particular -- that first activate the ubiquitin molecule. Then the third of these series creates a conjugation between the polyubiquitin chain and usually a terminal lysine.

The terminal lysine then targets the protein to be degraded for what is called a "26 proteasome complex" -- a multicatalytic, multisubunit type of protein. This complex contains various components, including a 20S core that is responsible for digesting the protein. The complex has 2 lids on each end of the core. These lids recognize the individual proteins that are candidates for degradation.

Once the protein goes through this cylinder, it is usually degraded into 6 to 9 amino acid components, which can either be recycled or presented through the antigen presentation system.

The complex is a very highly orchestrated system that regulates the half-life of proteins. Once again, these proteins are involved in cell-cycle regulation and cell survival. Proteins such as cyclins and cyclin-dependent kinase (CDK) inhibitors, for example, are regulated by this system.

In cancer cells, there is a dysregulation of this system. This dysregulation is important because it allows the proteasome to become a target for cancer cells, sparing normal cells. For example, if you use an inhibitor that in this case blocks the chymotryptic portion of the core -- in the case of a normal cell, the temporary or transient blockade of the proteasome -- the normal cell survives and goes on to recover.

On the other hand, if you temporarily block the proteasome in a cancer cell, the blockage creates a signaling chaos. This chaos, in turn, blocks cell-cycle progression and induces apoptosis. Thus there is a unique specificity here whereby the transient inhibition of the proteasome results in cancer cell death but spares normal cells. In that respect, the proteasome is a very unique target.

Clinical studies that validated that particular target and also allowed the FDA to approve it in the treatment of multiple myeloma. Bortezomib, the first prototype, is a single-target therapeutic agent with multiple downstream mechanisms.

Some of these mechanisms include stabilizing cell-cycle regulatory proteins such as p21 or p53, which would now be increased in cells and create a cell-cycle arrest. Bortezomib inhibits nuclear factor (NF)-kappa B activation, limits angiogenesis, and because of the accumulation now of proapoptotic molecules, induces apoptosis.

Bortezomib has multiple mechanisms of action that involve not only the tumor cell itself but also, most likely, its interaction with a tumor cell microenvironment. It's believed that the NF-kappa B transcription-regulating molecule is a critical target downstream of the proteasome. Normally when NF-kappa B is activated, it is transported to the nucleus to activate transcription. In order to activate NF-kappa B, you must first target the inhibitor of kappa B (I-kappa B), which is then phosphorylated, ubiquinated, and thereby again activates NF-kappa B.

Blocking the proteasomal degradation of I-kappa B, in turn, blocks the translocation of NF-kappa B and, therefore, downregulates critical proteins believed to be involved in survival and progression of the tumor. This sort of inhibition also affects molecules such as cell-adhesion molecules, which are critical for interaction and signaling with the tumor cell in the microenvironment, as well as regulation and production of certain cytokines.

Now let's focus a bit on the tumor microenvironment. Classically, when we target and develop drugs, we specifically analyze the effect of these drugs on the tumor cell itself. Similarly, in our investigation of drug-resistance mechanisms, we've also analyzed the tumor cell itself. I think this analysis is obviously important because there are intrinsic mechanisms, involved in both drug response and drug resistance, that exist in the cell itself.

But as I'll show you in a moment, these mechanisms are half the story. One interesting reason for success of the proteasome inhibitors, perhaps, is that they are prototypical drugs that target not only the tumor cell but also its microenvironment.

I'd like to introduce you to the concept of what we call "cell adhesion-mediated drug resistance." In other words, there's an interaction between the tumor cell and the normal microenvironment -- in this case the bone marrow for multiple myeloma. This interaction creates a sanctuary for tumor cell survival by both cell-adhesion molecules and the interaction between ligands in the environment and the cell-adhesion molecules on the surface of the tumor cell.

Cell adhesion-mediated drug resistance also occurs through soluble mediators such as interleukin-6 (IL-6). Interleukin-6 is known to be upregulated and increased in expression when a tumor cell interacts with the bone marrow stroma. Interleukin-6, as you know for myeloma, is a growth-factor stimulator and also prevents apoptosis induced by some drugs, including steroids, as well as Fas-induced apoptosis.

Thus this microenvironment, mediated by soluble factors as well as physical contact, can create a sanctuary in which tumor cells may survive. In addition, this microenvironment can prevent drug response and create a form of de novo drug resistance.

In 1999 we had first shown and reported that adhering multiple myeloma cell lines to an extracellular matrix, fibronectin, causes a drug-resistant phenotype. Since then, a number of cell lines have reportedly exhibited the same phenomenon. Also, there is a very multidrug-resistant phenotype that is very pleiotropic, showing resistance to anthracyclines, alkylating agents, as well as radiation. Imatinib (Gleevec) is associated with this phenomenon.

Even target-based therapy can be a candidate for this form of drug resistance, which has been principally described in hematopoietic cells, but more recently described, in solid tumors such as lung cancer, breast cancer, and ovarian cancer. This type of therapy, then, appears to be a fairly common form of de novo resistance.

This is just an example of how cell adhesion to fibronectin can prevent drug response -- in this case to doxorubicin or melphalan. This is looking at drug-induced apoptosis. As you can see, in adherent cells there is a protection.

This same phenomenon occurs in patients as well. This is a patient specimen with myeloma. If you expose the specimen ex vivo to a high dose of melphalan, you can see that the myeloma cells, which are labeled red, are undergoing apoptosis by the green staining of the nuclei using ApoTag.

If you take the same specimen, on the other hand, adhere it to fibronectin, and then expose it to melphalan, you can identify the myeloma cells, but they are surviving. By the mere adhesion to fibronectin through beta-1 integrins, therefore, you're creating a survival advantage.

In this case, 9 out of 10 patients exhibited this ex vivo phenomenon of taking patient specimens and exposing them to melphalan. You can see that the adhesion to fibronectin reduces the apoptotic events.

To elucidate mechanisms by which adhesion -- in this case in the environment -- is able to protect these cells from drug-induced apoptosis, we performed a gene-expression profile using microarray -- the Affymetrix system.

We found that 464 genes were repressed, but we're going to focus on genes that were actually increased. It turns out that 53 genes were increased over 2-fold, and 11 of these 53 genes appeared to be NF-kappa B regulated. This finding is important because we just said that the proteasome is very important in regulating activation of NF-kappa B.

This illustrates that if you attach a myeloma cell line to fibronectin -- and these are 5 different cell lines -- using an electrophoretic mobility shift assay (EMSA), you can see that NF-kappa B is activated in every case in which the cell line is attached to fibronectin. Thus, fibronectin appears to be a particularly important mediator of resistance, cell-cycle progression, and perhaps even disease progression.

These are just the 11 genes that I mentioned -- out of the 53 that were upregulated. There were some important genes involving apoptosis. c inhibitor of apoptosis (IAP)2 was upregulated by over 2-fold when the cells were attached to fibronectin, and appeared to be mediated, once again, by NF-kappa B activation.

There was something unique to the adhesion of these cells in terms of NF-kappa B activation. The mechanical or most classic heterodimer of NF-kappa B, when activated, for example, by cytokines such as tumor necrosis factor (TNF) alpha, is a heterodimer called p50/p65.

Although it has an NF-kappa B activation, fibronectin is unique in that the p50 portion or unit is activated, but it's now creating a heterodimer with RelB. The adhesion and the interaction between the microenvironment and the tumor cell is activating NF-kappa B by physical contact. Fibronectin is a unique heterodimer.

We felt that this was probably an important mechanism by which cell adhesion to fibronectin -- and we've gone on to show that this same phenomenon occurs with bone marrow stroma -- prevents apoptosis either due to drug-induced apoptosis or radiation. Most recently, we also illustrated that Fas-induced apoptosis was also blocked through at least one mechanism of RelB/p50 activation. In addition, we showed that the tumor microenvironment needs to be considered when we examine these myeloma cells -- or for that matter, any tumor. To isolate the tumor and examine the gene expression profile and expect that to represent the "true occurrence" is probably very unlikely. It is improbable that the environmental interaction between the tumor and cells within the environment or extracellular matrix can influence the gene expression profile.

The resistance profile associated with adhesion, as previously mentioned, is very pleiotropic. We're seeing resistance to alkylating agents, anthracyclines, radiation, and even immune-mediated mechanisms of death, including Fas-induced apoptosis. We have seen the rare drug that is not blocked by adhesion, but we had never seen a drug that was actually more active with cell adhesion until we studied bortezomib.

These are myeloma cells that are adhered to or were in suspension and then exposed to the bortezomib molecule. Look at the potency of the nanomolars. In every case, in a nice dose-response way, bortezomib was more active in cells that were adherent. Thus the drug probably works not only at the tumor cell level but also in the environment. Perhaps the upregulation of NF-kappa B, which appears to be a critical target of the proteasome inhibitors, may actually make these cells more sensitive to the bortezomib proteasome inhibitor.

In conclusion, we've shown you that adhesion to extracellular matrices, including fibronectin as well as bone marrow stroma, creates an antiapoptotic environment that is a sanctuary, if you will. These cells can survive exposure to various drugs. We believe that at least 1 mechanism is NF-kappa B dependent and that the proteasome inhibitor bortezomib may eliminate this form of resistance. In so doing, bortezomib acts not only at the tumor cell level, but is also interacting at the microenvironmental level.

Others have shown that beyond just the hematopoietic tumors, bortezomib can interact with various other chemotherapy agents, both in in vitro and xenograft models -- particularly of colon, lung, and prostate cell lines -- showing a synergism.

We believe that bortezomib, then, is a candidate not only as a single agent but also in combination with various other agents. In fact, bortezomib may block cell adhesion-mediated drug resistance, thereby increasing the efficacy of some of the classic agents that we are using -- which are affected by the microenvironment -- reducing dose response and enhancing drug resistance.

There are a number of ongoing clinical trials involving this compound. As I just mentioned, the indication for myeloma was approved by the FDA in May of 2003, and I believe there's a great deal of excitement about using this sort of agent in solid tumors as well.