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Spring 2004 Volume 5, Issue 10:
Drug Resistance: Overcoming Multiple Myeloma Drug Resistance by Inhibiting the Proteasome
By Robert Orlowski, MD, PhD
There are many difficult hurdles to overcome in the treatment of multiple myeloma, but one of the most important is a phenomenon called drug resistance.
04.26.04


There are many difficult hurdles to overcome in the treatment of multiple myeloma, but one of the most important is a phenomenon called drug resistance (recently reviewed in (1)). Myeloma cells that have this property can evade the effects of many chemotherapy drugs, allowing them to survive and even flourish in the face of such treatment. The impact that such drug resistance can have on the outcome of therapy may be seen in many different ways. Some patients have disease that, when it is first treated with chemotherapy, does not respond well, and occasionally may even progress, which is sometimes called primary refractory disease. If this happens, patients are usually switched to a different chemotherapy regimen, which hopefully is more effective. Other patients have disease that responds well and may even enter a complete remission, but after some period of time the disease relapses, and they need additional therapy. This usually occurs because a small fraction of the original myeloma cells were resistant to treatment, and once the majority that were sensitive were killed by chemotherapy, the remaining resistant cells had room and space to grow. Finally, many patients receive several different treatments for their disease, and may find that each successive regimen results in a briefer, and less dramatic benefit. One reason that this occurs is that some of the resistance pathways are active against several drugs, and therefore myeloma cells may survive therapy with one agent because a previous therapy activated a resistance mechanism that blocks the action of both. Fortunately, there are new ways emerging to overcome such drug resistance that may improve the effectiveness of myeloma therapy, but first let’s look at the molecular mechanisms by which this whole process occurs.

Inherited Chemoresistance
In thinking about the mechanisms of chemotherapy resistance important in myeloma, it is convenient to divide them up into several categories. The first is inherited chemoresistance, which is something that is present right from the beginning in the very first myeloma cell, and is passed down to all of the cells that arise later. Sometimes this genetic change may even contribute to the process of cancer formation itself. An example of this is the so-called Bcl-2 protein, which is expressed at higher levels than normal in plasma cells from many patients with myeloma. This protein is usually found in the membrane of mitochondria, which are the energy-generating organelles of cells. One of the ways that chemotherapy and radiation kills cells is by damaging mitochondria and inducing them to release a protein called cytochrome c into the cytoplasm, which then activates a process known as apoptosis, or programmed cell death. Bcl-2 is found in the membrane of mitochondria, and is thought to work against apoptosis by preventing the release of cytochrome c. Cells that overexpress Bcl-2 are resistant to a variety of therapies used in multiple myeloma, including anthracyclines like doxorubicin, alkylating agents like melphalan, and also steroids like dexamethasone. Such overexpression can account for the resistance of myeloma cells to the first therapy that they receive.

Acquired Chemoresistance
Another mechanism of resistance can be called acquired chemoresistance, because it is not present in every cell from the beginning, but appears in cells only after therapy has begun. An example of this is the so-called P-glycoprotein, which is not often found in myeloma cells initially, but after some types of chemotherapy its expression increases, and may be seen eventually in all plasma cells. The P-glycoprotein has a useful normal function, which is to recognize toxic chemicals in the cell and, from its position in the cell membrane, to pump them out, thereby saving the cell from damage. Unfortunately, many of the chemotherapy drugs used against multiple myeloma are also recognized as being toxins by this pump. By removing them from myeloma cells, the P-glycoprotein reduces the chances that the drugs will have their intended effect. Cells that overexpress P-glycoprotein are resistant to drugs such as doxorubicin and vincristine. Such overexpression can account for the ability of myeloma cells to resist treatments that they haven’t previously been exposed to, because the P-glycoprotein acts against many drug classes.

Inducible Chemoresistance
One last mechanism can be called inducible chemoresistance, and is one of the more recently described ways that this process of resistance can occur. Chemotherapy drugs are typically thought of as only killing cancer cells, but in fact they have many effects, and sometimes can actually activate signals that promote the survival of cells. This can be a desired outcome, because it may decrease the toxicity of some drugs against normal cells, thereby minimizing side effects to the patient. However, it can also be harmful, because if it promotes the survival of cancer cells then the drugs have actually decreased their own effectiveness against the tumor. In multiple myeloma one of the most important of these mechanisms is the so-called nuclear factor kappa B (NF-kB). When activated, NF-kB opposes apoptosis in a number of ways, one of which is by inducing a family of proteins called IAPs, or inhibitors of apoptosis. These proteins are able to slow the process of cell death stimulated by chemotherapy and radiation by binding to proteins called caspases, which are the catalysts that carry out the cell death program. NF-kB is activated in cells treated with drugs that are active in multiple myeloma, such as doxorubicin and melphalan, and also by other treatments, such as radiation. Since NF-kB is found in all myeloma cells, it can contribute to resistance to chemotherapy at every stage of the disease process.

Velcade®
The approval in May of 2003 of the drug bortezomib (Velcade®; formerly known as PS-341) by the Food and Drug Administration after positive results against multiple myeloma in Phase-I (2) and –II (3) clinical trials was an exciting development. This drug works by inhibiting the proteasome, which is a large complex in cells that is responsible for removing damaged proteins, and also proteins that have already served their purpose for the cell and are no longer needed (reviewed in (4)). Bortezomib, made by Millennium Pharmaceuticals, was approved for treatment of patients who have had at least two prior therapies, with progression of that disease on the last of these. Its ability to induce a complete remission in 10% of patients, and either a complete or partial response in 27%, was very encouraging (3). In addition, the drug was able to double the time to disease progression in patients with multiple myeloma compared to whatever had been their previous therapy. This is different than the usual trend with additional chemotherapy regimens for briefer durations of benefit, as described above. Perhaps most exciting about this drug, however, are studies that show bortezomib can help to overcome all of the mechanisms of chemotherapy resistance described above.

How Velcade® Counteracts Drug Resistance
Laboratory studies of cells that overexpress the Bcl-2 survival protein showed that exposure to bortezomib resulted in both phosphorylation of this protein and also cleavage into small fragments (5). This reduces the Bcl-2 level in cells, and likely makes them more susceptible to chemotherapy, which accounts in part for the ability of bortezomib to induce apoptosis independent of Bcl-2 status. With regard to the P-glycoprotein pump, studies have shown that function of the proteasome is needed for normal P-glycoprotein to be made (6, 7). When the proteasome is inhibited, abnormal immature forms of this protein accumulate which cannot pump chemotherapy drugs from the inside to the outside of cells. Since this results in greater levels of these drugs inside cells, it could enhance the ability of these drugs to kill myeloma cells. Finally, in terms of NF-kB, proteasome inhibitors work at several steps of this pathway, and block both the ability to make some parts of the NF-kB transcription factor (8), as well as its ability to move into the nucleus, where it activates proteins such as IAPs that block apoptosis. Based on just these findings alone, there would be excitement about the possibility that bortezomib could enhance the anti-tumor efficacy of standard chemotherapy drugs used in myeloma

Drug Synergy
Even more optimistic are laboratory studies looking at the ability of combinations of drugs including bortezomib to kill myeloma cells. In the first pioneering work that showed the promise of bortezomib against myeloma in the laboratory (9), investigators were able to show that the addition of steroids caused more cell death than either of the two drugs by themselves. This is called drug synergy, and is not always found between drugs, because some combinations will actually antagonize each other and cause less cell death, possibly compromising their clinical efficacy if they were tried in patients. Also, cells that had previously been resistant to steroids were once again able to be killed by these drugs when they were added in combination with bortezomib. Other drugs that are used in patients with multiple myeloma have been tested with similar results (10-12), showing synergistic cell killing, and also the ability to kill previously drug-resistant cells. These studies included agents such as melphalan, doxorubicin, and immunomodulatory analogs of thalidomide, and strongly supported the testing of such combinations in patients with multiple myeloma.

Velcade® Plus Melphalan
Based in part on the laboratory studies described above, several drug combinations either are being tested, or already have been, with a focus on myeloma, one of which is the combination of melphalan and bortezomib (13). Melphalan is also called Alkeran®, and is made by Celgene Corporation. In the most recent update of these results, presented at the December meeting of the American Society of Hematology (ASH), Yang and colleagues showed the outcome in the first 15 patients treated. From a side effect standpoint patients tolerated the drugs well, probably due in large part to the fact that the first dose level used melphalan at 0.025 mg/kg, which is one-tenth the dose used in the melphalan/prednisone combination, and bortezomib was used at 0.70 mg/m2/dose, or about one-half of the approved dose of 1.30 mg/m2. Ten patients had responses, including five with partial responses, meaning that there was a 50% decrease in measurable disease burden, and some of these included patients who had previously received melphalan.

Velcade® Plus Thalidomide
Another combination that has been studied is that of thalidomide and bortezomib (14). Thalidomide is also called Thalomid®, and like melphalan is also made by Celgene Corporation. At the ASH meeting last December results from the first 56 patients treated were presented, all of whom had relapsed after transplantation, and 81% of whom had previously received thalidomide, to which their disease had become resistant. Zangari and colleagues noted that the combination was well-tolerated, and while peripheral neuropathy was a frequent event, it generally was mild to moderate, and not severe. This is important because peripheral neuropathy is a known complication of both thalidomide and bortezomib by themselves, and one concern about combining them would be that patients would have even more of this problem. Neuropathy is caused by damage to the nerves and can present in many different ways, including numbness, tingling, burning, and pain, especially in the extremities, and sometimes can cause weakness and even impair the ability to perform activities of daily living. If such symptoms occur patients and their healthcare providers generally choose either to decrease the doses of the responsible drugs, or in more severe cases to stop the drugs altogether. There are also supportive care measures that can be used, such as supplementation with vitamins like B-complex, including B6, vitamin E, and folic acid, and also some prescription medications, like gabapentin (Neurontin) and Elavil. Neuropathy can improve over time once the drugs causing it are stopped, but the time course can be very long, and may take weeks to many months. Also, since nerve tissue does not regenerate or heal well, some patients may be left with a residual, permanent neuropathy. In addition to these findings on neuropathy from this study, more impressive was the fact that 22% of patients had a complete response, and 57% had either a complete or partial response. Such responses occurred even in patients whose myeloma had deletion of chromosome 13, which is usually a poor prognostic sign that predicts a worse response to most therapies than patients without this cytogenetic abnormality. Finally, the response rate was reported to be the same in patients receiving bortezomib at 1.0 mg/m2/dose and the lowest dose of thalidomide, 50 mg per day, compared with those receiving higher doses up to 200 mg of thalidomide daily. This is important because it suggests that it might be possible to reduce the doses of the drugs given to patients with comparable, or maybe even enhanced efficacy, and such reductions will make the drugs better tolerated.

Velcade® Plus DOXIL®
The last two-drug combination that was presented at ASH used bortezomib and pegylated, liposomal doxorubicin (15). This drug, better known as Doxil®, is made by Tibotec Therapeutics, and is a different preparation of the standard drug doxorubicin. Pegylated liposomes are lipid bilayers similar to cell membranes, and the doxorubicin is contained inside these structures. There are several advantages to this formulation compared with regular doxorubicin. One of these is that because the drug is released very slowly from the liposomes, it can be given intravenously over one hour, but stays in the body over a long period of time, similar to the effect of a continuous infusion of doxorubicin like many patients receive with the standard “VAD” regimen. Because of this, patients do not need either a central line for these infusions of doxorubicin or an infusion pump, and instead can receive Doxil® through a regular peripheral intravenous line. Also, the drug appears to be less toxic to the heart than standard doxorubicin which, if given at a cumulative dose in excess of 550 mg/m2, can cause a so-called cardiomyopathy, with decreased heart function that in severe forms causes congestive heart failure. In this study, which was done at our institution, bortezomib was given as an intravenous bolus dose on days 1, 4, 8, and 11 of a 21-day cycle in a range of doses, though the final study recommendation will be to start with 1.3 mg/m2/dose. Doxil® was given on day 4 about one hour after the bortezomib at a fixed dose of 30 mg/m2. Patients tolerated the combination well, and the side effects were those that would be expected if either drug were used alone. Side effects that were seen in at least 25% of patients included fatigue, thrombocytopenia (low platelet count), nausea, constipation, anemia, neutropenia (low white cell count), decreased appetite, diarrhea, neuropathy, and mucositis, or mouth sores. Responses with at least a 50% reduction in disease burden were seen in 73% of patients, with 36% having a complete response. Of particular interest is that there were 13 myeloma patients on the study who had previously received either standard doxorubicin or Doxil® itself, and whose disease had either progressed despite this therapy, or at best not responded and remained stable. Out of these 13 there were complete responses in 5 and partial responses in 3, suggesting that bortezomib was indeed able to reverse resistance to doxorubicin in at least some of these patients. Also encouraging is the fact that of the two patients who went on to transplant after this treatment, both were able to have good numbers of stem cells collected, suggesting that this combination does not damage stem cells and does not close the door to later transplantation.

Avenues for Further Research
All of these studies have so far been done only at single institutions, and the number of patients treated has been modest, so larger trials done at many centers will be needed to confirm these preliminary results. Several such studies are already being planned, and hopefully some of the results will be available soon. None of the combinations have yet been tested head-to-head in a randomized fashion, which is the best way to compare them directly, so it is not possible to know which is better, or if one is better for some patients while others may benefit from another. Other two-drug combinations based on bortezomib are being tested as well, including one trial of the thalidomide analog CC-5013, which is also known as Revlimid™, and made by Celgene Corporation. Finally, some investigators are looking at three-drug combinations, hoping that there would be a further improvement in efficacy. Thus, there certainly remains much work to be done in this area. Also, it should be pointed out that there are many other agents being studied that also may be able to overcome drug resistance in multiple myeloma. One good example is oblimersen, or Genasense™, being developed by Genta Incorporated, which is an anti-sense oligonucleotide that decreases levels of Bcl-2 protein.

However, there are many encouraging results that have been obtained so far from the bortezomib studies described above. The overall and complete response rates of some of the bortezomib-based combinations appear to be higher than what would be expected with either bortezomib alone, or with the standard chemotherapy agent alone. This supports the possibility that the drugs are acting synergistically to kill myeloma cells. Some patients with disease that was previously resistant to the standard chemotherapy have had significant responses, including many complete responses, to the combination of this same agent and bortezomib. This supports the possibility that bortezomib is able to overcome drug resistance. Also, the fact that some of these responses occurred at doses that were less than what would be considered standard is encouraging. If lower doses can be used, side effects will be decreased, and more people will hopefully benefit from therapy because fewer patients will need to stop treatment due to drug-related toxicities.

What This Means to You
What does all this mean for patients with myeloma, their families, and their health care providers? First of all, these combination regimens will be tested in additional trials, and by enrolling in these studies patients can have access to cutting-edge medicine using drugs that are known to have benefits against multiple myeloma. Only by doing such studies and collecting and analyzing the data will we be able to know which combinations work best and, possibly, which regimen is better for which type of myeloma patient. This may lead ultimately to the ability to predict ahead of time which therapy would be best for each patient as an individual. For those patients who do not have access to such trials, however, since all of these drugs are FDA-approved, it is possible to receive this therapy without enrolling on a clinical study. While these regimens will probably not be the cure for multiple myeloma, they may represent a new concept in the treatment of this disease. In the older paradigm, patients were treated with certain drugs, but once their disease no longer responded that drug was never reused, because the myeloma cells retained their drug resistance to that agent by passing those genetic characteristics on to their progeny. With the availability of drugs such as bortezomib, which seems able to overcome such resistance, this treatment paradigm may need to be changed. Patients whose disease was resistant to thalidomide or doxorubicin were able to have significant responses to these agents in combination with bortezomib, as noted above. Thus, it may now be possible to overcome the resistance of myeloma cells and recapture drug sensitivity, allowing the beneficial reuse of agents which would previously have not been considered options for patients because of prior exposure. As a result, the number of treatment options available to patients may be dramatically increased, which is the next best thing to a cure. Furthermore, the enhanced complete response rates suggests that such combinations will improve survival, and ultimately may form part of a multidisciplinary approach that will bring us closer to the cure for this disease.

References
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2. Orlowski, R. Z., Stinchcombe, T. E., Mitchell, B. S., Shea, T. C., Baldwin, A. S., Stahl, S., Adams, J., Esseltine, D. L., Elliott, P. J., Pien, C. S., Guerciolini, R., Anderson, J. K., Depcik-Smith, N. D., Bhagat, R., Lehman, M. J., Novick, S. C., O'Connor, O. A., and Soignet, S. L. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol, 20: 4420-4427, 2002.

3. Richardson, P. G., Barlogie, B., Berenson, J., Singhal, S., Jagannath, S., Irwin, D., Rajkumar, S. V., Srkalovic, G., Alsina, M., Alexanian, R., Siegel, D., Orlowski, R. Z., Kuter, D., Limentani, S. A., Lee, S., Hideshima, T., Esseltine, D.-L., Kauffman, M., Adams, J., Schenkein, D. P., and Anderson, K. C. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med, 348: 2609-2617, 2003.

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6. Loo, T. W. and Clarke, D. M. Superfolding of the partially unfolded core-glycosylated intermediate of human P-glycoprotein into the mature enzyme is promoted by substrate-induced transmembrane domain interactions. J Biol Chem, 273: 14671-14674, 1998.

7. Loo, T. W. and Clarke, D. M. The human multidrug resistance P-glycoprotein is inactive when its maturation is inhibited: Potential for a role in cancer chemotherapy. FASEB J, 13: 1724-1732, 1999.

8. Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell, 78: 773-785, 1994.

9. Hideshima, T., Richardson, P., Chauhan, D., Palombella, V. J., Elliott, P. J., Adams, J., and Anderson, K. C. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res, 61: 3071-3076, 2001.

10. Mitsiades, N., Mitsiades, C. S., Poulaki, V., Chauhan, D., Richardson, P. G., Hideshima, T., Munshi, N. C., Treon, S. P., and Anderson, K. C. Apoptotic signaling induced by immunomodulatory thalidomide analogs in human multiple myeloma cells: Therapeutic implications. Blood, 99: 4525-4530, 2002.

11. Ma, M. H., Yang, H. H., Parker, K. M., Manyak, S., Friedman, J. M., Altamirano, C. V., Wu, Z. Q., Borad, M. J., Frantzen, M., Roussos, E., Neeser, J., Mikail, A., Adams, J., Sjak-Shie, N., Vescio, R. A., and Berenson, J. R. The proteasome inhibitor PS-341 markedly enhances sensitivity of multiple myeloma tumor cells to chemotherapeutic agents. Clin Cancer Res, 9: 1136-1144, 2003.


12. Mitsiades, N., Mitsiades, C. S., Richardson, P. G., Poulaki, V., Tai, Y.-T., Chauhan, D., Fanourakis, G., Gu, X., Bailey, C., Joseph, M., Libermann, T. A., Schlossman, R., Munshi, N. C., Hideshima, T., and Anderson, K. C. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: Therapeutic applications. Blood, 101: 2377-2380, 2003.

13. Yang, H. H., Swift, R., Sadler, K., Vescio, R., Adams, J., Schenkein, D., and Berenson, J. R. A phase I/II trial of VELCADE and melphalan combination therapy for patients with relapsed or refractory multiple myeloma. Blood, 102: 235a, Abstract 826, 2003.

14. Zangari, M., Barlogie, B., Jacobson, J., Rasmussen, E., Burns, M., Kordsmeier, B., Shaughnessy, J. D., Anaissie, E. J., Thertulien, R., Fassas, A., Lee, C.-K., Schenkein, D., Zeldis, J. B., and Tricot, G. VTD regimen comprising Velcade (V) + thalidomide (T) and added dex (D) for non-responders to V + T effects a 57% PR rate among 56 patients with myeloma relapsing after autologous transplant. Blood, 102: 236a, Abstract 830, 2003.

15. Orlowski, R. Z., Voorhees, P. M., Garcia, R. A., Hall, M. D., Lehman, M. J., Johri, A., Humes, E., Adams, J., Esseltine, D. L., Gabriel, D. A., Shea, T. C., Van Deventer, H. W., Mitchell, B. S., and Dees, C. Phase I study of the proteasome inhibitor bortezomib in combination with pegylated liposomal doxorubicin in patients with refractory hematologic malignancies. Blood, 102: 449a, Abstract 1639, 2003.


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