Dr. Frank Zhan, MD, PhD and Dr. Guido Tricot, MD, PhD University of Iowa Interview Date: May 6, 2015 Summary The 14;16 and 14;20 myeloma translocations are rare - in about 6% of all patients. These patients can respond to therapy just like normal risk patients, but the duration of their remission can be much shorter. Dr. Frank Zhan, MD, PhD describes his research to search a genetic database to find 100 genes that may be affected in the 14;16 and 14;120 myeloma translocations. That work led to search a compound database to find 5 possible drug targets that would impact the genes that are changed by these translocations (c-MAF for 14;16 and MAFB for 14;20). After testing these in the lab, one compound stood out that could stop the growth of cancer cells for c-MAF and MAFB - Allsterpaullone. In addition, Drs. Zhan and Tricot found that expression of Notch2 is strongly regulated in this MAF subgroup. Notch2 is an important gene related to cancer stem cells, drug resistance and the bone marrow microenvironment. The proposed research would be to use Allsterpaullone and a new Notch2 antibody now in clinical trials to provide the same length of remission (and beyond!) for this high-risk patient group. The Myeloma Crowd Radio Show with Dr. Frank Zhan, MD, PhD and Dr. Guido Tricot, MD, PhD
Jenny: Welcome to today's episode of Myeloma Crowd Radio, a show that connects patients with myeloma researchers. I'm your host, Jenny Ahlstrom, and I'll be joined today by my myeloma friends and co-hosts including Jack Aiello, Gary Peterson, Pat Killingsworth, and Lizzy Smith. This is the sixth in a very important series featuring the Myeloma Crowd Research Initiative. Now, if you haven't already heard about the MCRI, educated patients are teaming up with myeloma researchers to find and fund ideas in myeloma that could have the greatest impact for the next generation of myeloma therapies. We decided to go after high-risk myeloma because these patients have very poor outcomes and very few working therapies. Ultimately, if we become relapse or refractory to current drugs out there, we also become high-risk, and what's been found is what works in high-risk will help low or standard risk patients as well. Here's what we've done so far. In February, we asked researchers around the world to submit their proposals and we received 36 high-quality letters of intent back. In March, that list was then scored by our Scientific Advisory Board who selected their top ten proposals, and in April and May, we're now holding Myeloma Crowd Radio Shows so you can be involved. We'd love for you to understand the proposals, so please listen in, ask questions, read the transcripts after the show, post it and share it with your friends and family. This is very critical work being done in myeloma. After the full proposals are submitted and the shows are complete, the Scientific Advisory Board and Myeloma Patient Advisory Board will together decide on a limited number to fund through patient-driven campaigns, and again, we will need your help to get the word out and share the really amazing work that's being done. Now, each myeloma patient has a whole community around them who want to help, but sometimes they don't know how. This will be a way that they can help you find a cure for your disease, so watch out for our upcoming announcement next week on how you can build a team and get started. We're very privileged to have with us today Dr. Frank Zhan of University of Iowa and joining him is Dr. Guido Tricot. Welcome, doctors!
Dr. Zhan: Hello! Good afternoon.
Dr. Tricot: Good afternoon.
Jenny: Thank you so much for participating and let me introduce you both. Dr. Frank Zhan is Professor of Internal Medicine in Hematology, Oncology and Blood and Marrow Transplantation at the University of Iowa. Dr. Zhan received his medical degree at Hunan Medical University in China and his PhD in Cancer Molecular Genetics there as well. He performed his post-doctorate work in both Kentucky and at the University of Arkansas for Medical Science. The long-term objective of Dr. Zhan's lab is to develop new therapies to overcome drug resistance and deplete cancer stem cells in myeloma. His work focuses on finding the genes that make myeloma cells resistant to chemo and then to use that knowledge to test new hypothesis in the lab and in mouse models. Dr. Guido Tricot is Director of the Bone Marrow Transplant Program at the Holden Cancer Center at the University of Iowa. Dr. Tricot was educated in Belgium and has served as the Director of Bone Marrow Transplant and Myeloma Program additionally for the Huntsman Cancer Institute, the Greenbaum Cancer Center at the University of Maryland and Indiana University. He was also the Director of Clinical Research at the University of Arkansas Myeloma Institute for Research and Therapy. Dr. Tricot has been treating myeloma patients for over 20 years. So again, welcome to you both. I think we will add another one of our co-hosts. Pat, are you on the line?
Pat: Hi, Jenny! Can you hear me?
Jenny: Yes we can. Thank you for joining.
Pat: You bet.
Jenny: Thank you for joining us as well, Jack. I think we would like to get started with some basic biology. Maybe, Dr. Tricot, you'd like to help us understand what happens when genes are translocated or what does that mean.
Dr. Tricot: Every gene is normally located on a certain chromosome. When a gene is translocated, it goes from one chromosome to another chromosome. By doing that, it basically jumps away from where it originally belongs to an area where it thinks it has more benefit to be there than in the original area. That's the basis of a translocation, so it's part of the chromosome that is jumping away from where it used to be to a new chromosome.
Jenny: It seems like the 14-gene is usually involved in myeloma. When you say you have these translocations, there's t(4;14), t(14;16), t(11;14), or t(14;20), why are they all involved in gene-14?
Dr. Tricot: 14q, the long arm of chromosome 14, is the place for the heavy chain of the immunoglobulin. You know myeloma is a disease where the plasma cells make abnormal M proteins which are derived from normal immunoglobulins, and an immunoglobulin has a heavy chain and a light chain. A heavy chain gene is on chromosome 14q, on the long arm of chromosome 14. That's why it's so frequently involved in myeloma translocations.
Jenny: Today, we're talking about specifically the t(14;16) and the t(14;20) translocations, correct?
Dr. Tricot: Yes.
Jenny: It's my understanding that when a translocation occurs that it changes certain or very specific genes. Can you explain what genes are affected by these translocations and then what those genes do?
Dr. Tricot: The reason why the important genes go to chromosome 14 is because the promoter of the gene for the heavy chain of myeloma cells is a very strong promoter. A promoter needs to be seen like an ignition on a motor. The motor by itself doesn't work. The gene is only transcribed when the promoter is active and when it ignites the gene to work. The promoter of the heavy chain gene in myeloma which makes all the immunoglobulin is extremely strong. And so, what the other genes are doing is they're jumping to that chromosome 14 so that they are now coming under the direction of a much stronger promoter, a much better igniter than what they were originally. Therefore, they're very overexpressed in that way. They have much more activity in that way than if they were at their original place.
Jenny: So what might have been contained before by regular processes are changed?
Dr. Tricot: It changed, yes. Those genes, they take advantage of the fact that the heavy chain promoter is such a strong promoter so that they now come under the direction of this very strong promoter, which they didn't have before. Therefore, they are much more expressed and much more of that abnormal protein is being made.
Jenny: On the Myeloma Crowd site, what we decided to do is to break up these different Facebook groups into gene-specific Facebook groups, so we have a general group and a family caregiver group which patients can join if they would like, but we also have gene-specific groups. Can you just give an overall assessment of why the knowledge of your own genetics of myeloma is so critical and how you determine what your features are?
Dr. Tricot: When you look under the microscope, all the myelomas look typically about the same. There's not a lot of variation, but when we look at gene expression profiles of patients with myeloma -- and we have done that on over 400 patients -- we saw that there are clearly distinct groups. The groups are mainly made by the fact that myeloma cells have more than 46 chromosomes which we call the hyperdiploid group or has translocations in it involving t(11;14), which is a Cyclin D1 and which is associated with good prognosis, the t(4;14) which is the MMSET translocation which is associated with poor prognosis, and t(14;16) and t(14;20) translocations, which also have poor prognosis. Although we have made a lot of progress in the treatment of myeloma, it's especially in those poor translocations that we still have quite a bit of problem getting the disease under control for long periods of time. Getting the disease under control is not that difficult, but keeping it under control for a long period of time, that's a totally different issue.
Jenny: These are just features where certain genes are -- so we have these features separated out in these Facebook groups. When different therapies are introduced for a specific kind, for example, del(17p) patients might be better off with bortezomib or something like that, then you can start to get to a more personalized therapy. You're finding this out, these genetic features, through the FISH and the gene expression profile test, or what other testing are you doing to find these translocations?
Dr. Tricot: That's exactly right, through the gene expression profiling and through the FISH analysis.
Jenny: Dr. Zhan, maybe you can explain the process that you went through to find or to discover what you would like to talk about today because I think the process that you're going through is really, really interesting.
Dr. Zhan: That's right. We have developed under this data seven genetic subtypes such as c-MAF, MAFB, Cyclin D1, Cyclin D3, and MMSET actuating translocations, and those are hyperdiploidy use gene expression profiling. So those indicate this deregulation of common pathways by gene orthologs, common gene signatures were found in cases with the c-MAF and MAFB. That's why we've classified c-MAF and MAFB as the same subgroup, but they are different translocations - c-MAF would come from t(14;16) and the MAFB due to the t(14;20). Then we identified 100 genes uniquely disregulated in MAF included in both the c-MAF and MAFB subgroup. We predicted compounds that could induce or reverse the gene expression changes based on the connector map. This Connect database is a comprehensive database including compounds in the micro-array data set. However, we only found that the drug named Allsterpaullone significantly inhibited myeloma cell growth with extremely high expression is c-MAF or MAFB. That’s the reason we came to our proposal, to use this Allsterpaullone to target specifically this type of myeloma disease. In addition, we also found that expression of Notch2 is strongly regulated in this MAF subgroup. Notch2 is an important gene related to cancer stem cells and drug resistance and also the microenvironment. So another drug we are using to target this subgroup is a Notch2 antibody. That’s the whole process of our project.
Jenny: That's a great summary. I'm going to ask you to dig a little deeper. This is a pretty complex stuff, so I want to make sure that patients understand it. So your first step was that you divided all your samples into seven different genetic subtypes. Each of those different subtypes deregulate different genetic pathways. Then you were focusing on just the (14;16) and the (14;20) translocations, and those affect a gene called c-MAF for the (14;16) and MAFB for the (14;20), right?
Dr. Zhan: That's right. That's correct.
Jenny: And then your second step was you try to look at all the genes that were deregulated for those two. And you found a hundred genes, right?
Dr. Zhan: Yes. We used the target of 100 significant genes. There are more than 100x expression in this subgroup, but for this inquiry we only chose the top 100 genes that are manifest.
Jenny: So you're looking through a massive database of genes and you're trying to find the genes that might be causing these problems.
Dr. Zhan: That is correct, yes.
Jenny: My way of saying it is very basic. And then you connected what you found with those genes to existing drugs or could be drugs to then try to match up a drug with those mistakes in the gene, right?
Dr. Zhan: Yes, so we used the 100 uniquely disregulated genes in these translocations of 14;16 and 14;20 into the Connector MAF database. In this database, it includes 1,200 compounds and a few thousand micro-array. So once we input our 100 unique genes, based on this goal for connectivity or enrichment, we can find which compound can reverse this MAF signaling pathway. Finally we found five compounds can do that function. But based on the bench work (in the lab), we found one compound can inhibit MAF signaling pathway and kill myeloma cells.
Jenny: So your next step was that you matched that compound database up against those genes and then you found five compounds that potentially could but then you only found really one compound that could reverse that bad signaling pathway, right?
Dr. Zhan: Exactly.
Jenny: And that is called Allsterpaullone? How do you say that? I don’t know how to say that word.
Dr. Zhan: Allsterpaullone.
Jenny: It seems like it's also called ALP, right?
Dr. Zhan: That's right.
Jenny: Can you explain, either one of you, what ALP does and how it changes that? Because when I was reading through your proposal you mentioned that these two, the (14;16) and the (14;20) that are c-MAF and MAFB, they are resistant to bortezomib. Since most of us are getting bortezomib as part of our treatment, can you explain what that gene is doing and then how drug is affecting it?
Dr. Zhan: Okay, I go first. So ALP is a small molecular compound which belongs to the class of organic compounds known as benzazepines. This organic compound contains a benzene ring fused to azepine ring. That’s the structure of this compound. The functions for this ALP are an inhibitor for cyclin dependent kinase and an WNT signaling pathway inhibitor. That is already known. We also know the translocation 14;16 and the 14;20 both the pathway of cyclin dependent and WNT signaling pathway are activated too. That is good evidence for using this drug to target these pathways in MM. Then about the resistance to Velcade, or bortezomib – We already know, actually not done by our group, from the published data, one group proved bortezomib can phospholate the MAF or MAFB. The good thing is that if bortezomib phospholates MAF or MAFB, can degrade these oncogenes or MAF/MAFB genes. Another aspect is this bortezomib can also activate MAF or MAFB downstream signaling. In my expectation, the later one plays a major role in MM disease. That may be the reason that bortezomib also activates MAF or MAFB downstream signaling. That’s why they (MM cells) are resistant to bortezomib.
Jenny: So you're saying bortezomib creates -- sorry, I'm not understanding it as well as I should. It's degrading the active signaling?
Dr. Tricot: You would expect that bortezomib would prevent the downstream signaling of MAFB and MAFB - the signal that does bad things. And you would like to have a drug that prevents these bad things from happening. But actually, when Frank did his analysis, instead of inhibiting this downstream signaling of MAFB and all the bad things, it actually in some ways activated this downstream signaling. That's why it doesn’t work very well. It works for a while but then it stops working.
Dr. Tricot: One of the basic problems in myeloma is that there are so many redundant pathways. You block one pathway, it goes through another pathway. You think you're doing well but actually the only thing you're doing is, yes, you block one specific pathway but now it starts to fire through other pathways which will lead to the same end result, meaning resistance to the drug.
Jenny: So you need not just one blocker. You need a whole team of blockers.
Dr. Tricot: Exactly. There is only one disease in hematologic malignancies which can do with a single blocker and that's chronic myeloid leukemia (CML) because everything has to go through the Philadelphia chromosome but in all the other diseases, it is much more complex and a single drug has never worked in any of those diseases.
Jenny: So what would ALP do with the MAF and the MAFB?
Dr. Tricot: ALP is a cyclin inhibitor. That means that the cyclins are necessary for the cells to grow and proliferate. If you inhibit that, then the cells cannot grow and proliferate anymore.
Jenny: Would you still be able to use it with bortezomib?
Dr. Tricot: That's one of the things we would like to test whether if we combine this ALP with bortezomib whether we can make the cells sensitive again to bortezomib. We don’t know that yet but that's one of the things we absolutely want to see.
Jenny: Have you seen the same effect in carfilzomib that you see with bortezomib in terms of that it's activating it in a negative way?
Dr. Tricot: Yes, they are all of the same class of drugs - the proteasome inhibitors. And they all work the same way.
Jenny: So no matter what proteasome inhibitor you use, whether it's bortezomib or carfilzomib, it would have the same outcome?
Dr. Tricot: Yes.
Jenny: Well, that's not good because that's like a standard therapy that's used in most everyone's treatment.
Dr. Tricot: Yes.
Jenny: It inhibits proliferation and then it allows the other drugs to work. And then, Dr. Zhan, you also mentioned that there was another aspect to this that you found a Notch2 pathway. Can you both elaborate on that?
Dr. Zhan: Yes. I go first. The Notch signaling pathway is an important signaling pathway in myeloma stem cells. This pathway is widely studied in other cancers. Even in myeloma, inhibition of notch signaling can induce cell apoptosis to kill myeloma cells. We found a drug to specifically target Notch2. In our prospoal we found that Notch2, one of the Notch receptors (there is Notch1, Notch2, Notch3 and Notch4), is uniquely upregulated in this MF (c-MAF/MAFB) subgroup of myeloma. Our idea is if we can use this Notch2 specific inhibitor or antibody then we can personalize to treat these translocations of 14;16 and 14;20. Currently, the Notch 2 antibody is available in clinical trial in Phase I and II for pancreatic and lung cancers and other solid tumors. That is why we would like to bring this Notch2 antibody into this unique subtype of aggressive myeloma disease.
Jenny: So Notch2 is a signaling pathway and you found this -- what's the difference between an antibody versus an inhibitor?
Dr. Tricot: There are many ways to deal with a receptor. One is you can use a small molecule that inhibits the function or you can use an antibody that blocks the function. Ultimately, the end result is about the same. But in general, if your receptor is not mutated, if there are no changes in the receptor, you're better off with an antibody than with a small molecule inhibitor. There is another group of drugs which he talked about, the gamma secretase inhibitors which are not selective. You have to remember that these pathways are not only important in cancer but they are also important in normal blood formation cells and in the gut cells that we make every day and normal stem cells that we have in our body everywhere. So the more selective you can be and the more you can only inhibit what you really want to inhibit and not inhibit things that can only be detrimental and have no benefit to myeloma, the better off you're going to be. That's why this antibody which is very selective for Notch2 will be a better approach than generally inhibiting Notch as a whole.
Jenny: Because it sounds like there's Notch1, Notch2, Notch3, Notch4. You might stop something that you don’t want to stop.
Dr. Tricot: Exactly. You can imagine that if you block all Notch that you will have problems with your normal blood formation, the normal hematopoiesis and also the normal cells of the gut, the stem cells of the gut. You see that if you give those gamma secretase inhibitors, one of the common side effects is that these patients have a lot of diarrhea because you mess around with their gut stem cells.
Jenny: Interesting. I think it's the challenge of multiple myeloma because there's not one single target that you're going after. You're going after all these targets. So in trying to find drugs that are effective, you can either shut everything down which would be dangerous or you're trying to be very specific which is what you're trying to do in this proposal. You're trying to say for these patients that have this specific translocation, this might be their silver bullet or their target that might work for them specifically.
Dr. Tricot: There is something in cancer that they call oncogenic addiction. That means that the cancer cells become addictive to a certain oncogene. If you can block that oncogene that makes the cells survive and grow and do well, you can basically prevent this from growing. While the normal cells, they don’t have this addiction. They depend on many different pathways. They cannot be inhibited by just a single intervention or a few interventions.
Jenny: So you're trying to isolate those pathways that are most effective only for the cancer cells?
Dr. Tricot: What we want to do is try to isolate the pathways which are majorly upregulated in (14;16) and (14;20) much more than in normal cells. So the more they are upregulated and the more they are different from normal cells, the better we like it. That's how we can selectively go after the cancer cells.
Jenny: That's what flags it for you.
Dr. Tricot: Yes.
Jenny: Can you explain what your next step is then? If you've gone through the database, you found a compound and now you're going to try it in multiple myeloma, can you explain what happens next?
Dr. Zhan: Yes. In our preliminary data, we tested this compound in myeloma cell lines. So these cell lines are including these translocations 14;16 and 14;20. We found this drug ALP can uniquely kill myeloma cells with these translocations but are low in effectivity for myeloma cells without the 14;16 or 14;20 translocations. So after that in our proposal we will try to do some in vitro and we will study this using primary myeloma samples in vivo because this is the key to translate this drug into the clinic. We propose to collect 5 primary myeloma samples with or without these translocations (14;16 and 14;20) and use this drug to do in vitro co-culture of myeloma cells with stroma cells in this in vitro model. Then we will also use the mouse model to mimic human myeloma disease (for in vivo study). We inject this tumor cell into fetal bone into the mice so from this case we can get an idea if this drug is specifically targeting these translocations in primary myeloma samples. We will test what this is doing for ALP but we will also test the Notch2 antibody for this specific translocation samples. Maybe we will also do a combination with these two drugs with others such as bortezomib or iMiDs we propose in the first aim. We know some targets of C-MAF or MAFB if we cannot directly target these two oncogenes. We tried to use the new technology because the next generation sequencing is a really powerful tool that can be used to identify C-MAF and MAFB direct targets. If we find some novel targets then we can also see if these targets can be translated to the clinic again. That is our second aim in this proposal.
Jenny: Well, let me ask you a question about these different compounds. I know these compounds are in this database and you're matching them up. Just out of curiosity, who is developing these compounds? Because you're testing samples and it sounds like your first step is to do it in the lab in vitro in a dish and then try it in actual live mouse model as a second step and then maybe with or without bortezomib and with or without this Notch2 inhibitor. But I'm just curious where these compounds come from. You said it was an organic compound, but I just think it's amazing that these compounds exist in a database somewhere that you can use to test.
Dr. Zhan: Actually we bought this ALP compound from Sigma. We bough the Notch2 antibody from OcoMed, a pharmaceutical company because they provide this compound for clinical trials. We are trying to contract with them to test in our model.
Jenny: This other drug that the Notch2 signaling pathway is, what's the name of that?
Dr. Zhan: Tarextumab. Jenny: And you said it was being used in Phase I and Phase II trials for pancreatic and lung cancer already, correct?
Dr. Zhan: Yes. That's correct.
Dr. Tricot: And you have to remember that when the drug companies develop a drug, that they are always trying to go for the large markets. Compared to other solid tumors like lung cancer and breast cancer and prostate cancer, myeloma is relatively small fish. So they will always try to see whether their drugs work initially in tumors that are much more frequent than myeloma.
Jenny: And then I think you layer on top of that, a small percentage of myeloma patients have the (14;16) and (14;20) translocations.
Dr. Tricot: About 6% of all myeloma patients have these translocations. But those are actually the patients that are doing the worst. When we look at our overall profile about 83% of our patients are doing very well with the current treatments and about 17% are not doing so well and actually are not doing too much better than 10 or 15 years ago. It's especially those who have these translocations, the (4;14) and the (14;16) and (14;20) translocations and the deletion 17q. Those are the patients that we still need to do a lot better for and make sure that their prognosis becomes as good as what we see in other patients.
Jenny: I think that's one of the reasons why this MCRI project exists is because the funding might not come from the large pharma companies to go after a small target. But if you can find a cure for multiple myeloma in one type, then it might not be the same as other translocations or other genetic features. But you can say that you've solved one piece of the puzzle.
Dr. Tricot: Yes. Then again there will be other cancers that have the same 100 genes that are differentially expressed compared to others and in those it should also work.
Jenny: I have a question about that also because you look at some patients like I know that Mike Katz just recently passed away and he had multiple myeloma and he also had colon cancer. Do the same genetic deregulation of the genetic pathways exist in your mind for both types of cancers? Could he have had the same genetic errors I guess?
Dr. Tricot: There is no direct evidence that the same important genes for myeloma will also affect colon cancer. But it's not sure that when you would look at cancer stem cells and colon cancer and in myeloma that those genes may be much more comparable than if you look at the bulk of cells. What we typically do is we take a sample of a patient with myeloma who is just diagnosed and the bone marrow is almost full of myeloma cells, and it's very easy to find the gene expression profile. But we also know that about 80% of the patients go into complete remission with appropriate treatments. That means that 80% of the patients you eradicate more than 99.9% of all the myeloma cells in the body, meaning that those patients have very sensitive cells to chemotherapy and that there's only a small percentage of the cells that is much more drug resistant. Instead of looking at the gene expression profiles in the beginning like most people do, we have been always more interested in looking at the gene expression profiles of cells that have survived our intensive treatments and by definition or drug resistant. We use our chemotherapies as a way to select for the most drug resistant cells. We think that that is a much more informative approach than to look at what the gene expression profile shows at the time of diagnosis.
Jenny: Because you're just determining what's surviving the chemo.
Dr. Tricot: Exactly, exactly. What are the cells that are surviving and what do the genes show us and is there something in common between all those patients that have cells surviving the treatments. If there is something in common, how can we attack that and make those cells go away? Now research has basically shown that the major problem in myeloma is the myeloma stem cells, which are the mother cells. We used to think that in cancer, every cancer cell is about the same and can cause the disease to relapse and to grow. We now know that there is only a very small fraction of cancer cells that can do that, and those are the cancer stem cells or the mother cells; while the daughter cells, they have a limited life span and after a while they will just die and they cannot renew. They cannot replenish themselves. It's only the stem cells that can do that.
Jenny: So you're trying to target the source basically?
Dr. Tricot: Exactly. That's why we are so interested in Notch because Notch is a typical gene that is upregulated in all types of stem cells, whether it's cancer stem cells or hematopoietic stem cells or embryonic stem cells, it's upregulated in all those stem cells. But we want to go for the notch that is the most relevant to myeloma and not relevant to other diseases and not normal cells.
Jenny: How do you test for an upregulation of Notch2? What test shows that?
Dr. Tricot: You look at the amount of protein of Notch2 in the myeloma cells that are the (14;16) translocation or (14;20) and then also in the ones that don’t have those. You see that you see much more Notch2 protein in those cells that have the (14;16) and (14;20) translocations. There are people in the Netherlands who have taken myeloma cell lines that are very low expression of Notch2 and they introduce the MAF gene so that the MAF gene was overexpressed in those cells. Although originally it was low, now after transfection, there was high expression of the MAF genes. What they saw is as soon as they introduced that MAF gene, Notch2 goes way up.
Jenny: So that was a clear result.
Dr. Tricot: Yes. And then Dr. Zhan has also shown that if you take the opposite, you have cells that have high expression of MAFB and you block Notch2 in those cells because the high MAFB was high notch that those cells are dying. He has shown that in vitro in cells.
Dr. Zhan: Originally we discovered this Notch2 based on our gene expression profiling from almost 500 samples. For this specific subgroup for C-MAF and MAFB, we found that Notch2 is at least two fold higher than other subtypes of myeloma. That’s why we decided to target it for this subtype.
Dr. Tricot: Again, remember that the more the abnormal cells are different from normal cells, the better we like it and the better you can specifically target and the more differential effect you will see on the cancer cells versus the normal cells.
Jenny: Just because they're more specifically defined?
Dr. Tricot: Well, they have higher expression of Notch2 than normal cells. So if you block Notch2, they will be much more affected by that than in cells that have also Notch2 but at a much lower level.
Jenny: Do a lot of patients that have (14;16) or (14;20) have multiple genetic issues? Because sometimes I know that for deletion 17, patients will pick up that deletion over time. Is that the same with (14;16) and (14;20) or do you have it and then just keep it?
Dr. Tricot: If you don’t have it, you never get it. If you have it, you have it from the beginning in contrast to deletion 17 which you can acquire.
Jenny: Okay, perfect. So Gary and Jack, do you have any questions? I have several more but I want to allow you time as well.
Jack: This is Jack. I have several questions but, boy, this has really been educational. I really appreciate the doctors trying to dumb it down for me.
Jenny: Yes, same for me.
Jack: Just to clarify for my sake, I think I've read that t14 and translocation (14;16) and (14;20) each occur in about 1% of myeloma patients. Is that true?
Dr. Tricot: That's underestimating it. In our studies, we found that 6% of the patients have either one of those translocations.
Dr. Zhan: So that's about 3% for each.
Dr. Tricot: Yes. One percent of each is an underestimation.
Jack: So 3% of each, okay. Do all of those t(14;16) and (14;20) have this oncogene CMAF or MAFB?
Dr. Tricot: Yes.
Jack: Do those oncogenes get expressed? And you might have answered this. I apologize. Do they get expressed in other normal cells as well?
Dr. Tricot: MAFB is expressed in other cells too but at a much lower level.
Jack: So hopefully, that giving out, for example, which inhibits the expression won't have significant undesirable side effects?
Dr. Tricot: No. When Dr. Zhan looked at the cell lines that had the MAF translocations, those were killed but all the other myeloma cell lines that didn’t have the MAF translocations, those were unaffected. So we also think that the normal cells are going to be totally unaffected by this.
Dr. Zhan: And overexpression of MAF or MAFB in myeloma is over 10,000 times higher level than without those translocations. That is why we believe that there may be a gene onco-addiction for this specific oncogene. In our preliminary data our drug ALP only killed the myeloma cells with translocations 14;16 and 14;20 but not those without the translocations. Our prediction is the side effects should be very, very low in normal cells.
Jack: What about in other myeloma cells, do those c-MAF and MAFB oncogenes appear and don’t have translocations?
Dr. Zhan: One report from the NIH a few years ago said that the 4;14 translocation with MMSET can actually upregulate in C-MAF, but this upregulation is much lower than in the original translocation by the 14;16 and 14;20 translocations. They are at least 100 fold.
Dr. Tricot: Again, this comes back to what I was trying to explain in the beginning that if you have this translocation, you now bring this gene under a different promoter, a much stronger promoter, a better igniter to get this gene going than what it is originally. If you don’t have the translocation, you can still have increased levels of c-MAF but at a completely different level than when you have this translocation. Again, the difference is about 10,000 times.
Jack: Got it. I understand. Those are my questions and I really appreciate it. I'm learning as you are discussing and it's really informative.
Dr. Zhan: Thank you.
Jenny: Before Pat asks his questions, I have one more follow-up based on what you just said. Can patients have the (14;16) or 14;20 translocation on some of their myeloma cells but not on all or do they have it on all cells? So if you go after it with this target, it would all be killed or would you need some kind of companion thing to go after, let's say, this hits 50% and -- anyway, does that make sense?
Dr. Tricot: All the cells in patients who have the c-MAF translocation, all the myeloma cells have this translocation. But your second question, do you think that you can kill all the myeloma cells with a single agent? I think that's quite naïve. I think we need to have combinations. That's why we are trying to combine this alsterpaullone with the Notch2 inhibitor and probably with other drugs to see whether these combinations can kill all the cells. But the single agent killing all the cells, I think that doesn’t happen.
Jenny: Just because there are too many ways of myeloma getting around it.
Dr. Tricot: Exactly. There are always too many ways to fire through other pathways and getting around the block. We put a block up and then they will go around and it still get to the same end point.
Jenny: Okay. Perfect. Pat, did you have questions?
Pat: I do. Thanks, Jenny. Is there anyone better at explaining this technical material than Dr. Tricot?
Jenny: I think it's great.
Pat: Doctor, you're the best. So this was a good lead in for my questions because I wondered, first of all, it's great just imagine you're one of these patients and you know that Velcade or Kyprolis isn’t going to work and so why even give it to them and just knowing that is a service to a patient's quality of life. But like Jenny said unfortunately that takes a whole class of drugs out of the mix. So you get this figured out and you successfully attack this particular – or this is working. So I guess the million dollar question is how long does it work? What happens in the three, four or five year? Does it relapse just like someone responding to IMIDs and then the IMIDs stop working?
Dr. Tricot: Obviously, without doing a clinical study we cannot answer this. But before we can do a clinical study we need to have more convincing data that this works indeed not only in the tube but also in the mouse model where you have the normal microenvironment of a human system where a lot of our drug resistance is caused by the microenvironment, by the soil where the myeloma cells grow in. If you don’t take care of the soil, you cannot get the disease eradicated. We want to do this also in vivo in mouse models where you have a normal human fetal bone implanted with normal human microenvironment. You inject a patient's myeloma cells in there and then you give the drugs to the mice and you see where you can kill this myeloma. When we can do that, then we are ready for clinical trials.
Pat: That's great. In your experience, when you do that, how well does the mouse model really translate to the clinical trial? I know patients get excited, in vitro they get excited about the pre-clinical stuff and then there's disappointment or trials that never even get off the ground. So in your experience, how often does this work? So things are working as you expect in mice models. You guys are incredibly smart guys. Let's assume that's the case. So what are the odds that will translate to humans and this thing might actually work?
Dr. Tricot: That probability is not higher than about 10%.
Dr. Tricot: Yes. We have cured myeloma in the mice a hundred times over. But we have not cured myeloma in the large majority of patients with myeloma. We can make them live long. There are some with good prognosis that live longer than 15 years and not probably cured but that's still a minority. The proof is always in treating patients and seeing how long it will take for the disease to come back.
Pat: Sure. What an incredibly honest and yet disconcerting, disappointing answer. Ten percent is still -- come on, I can't believe it's only 10% for you guys but thanks for being so modest and honest. It sure sounds like you're on the right track.
Dr. Tricot: When I say 10%, I base that on it's 10% of all the compounds that are developed by the pharmaceutical companies and that have done all these same studies in the test tube and then subsequently the mice, and only one of out of ten product that they think looks very promising is actually when you do the human studies is very promising.
Pat: And you're probably just like patients. You're always expecting to be in the upper half or the upper 10%, right? So you wouldn’t be doing this if you didn’t think it's going to work just like a patient wouldn’t try a certain therapy working with their doctor hoping and expecting that it wasn’t going to work for them. But we'll keep our fingers crossed.
Dr. Tricot: So we all hope that we live on Lake Wobegon where every child is more than average.
Pat: Thanks a lot, doctor. Thank you, Jenny.
Dr. Tricot: Thank you.
Jenny: Thank you so much, Pat, for your questions. Well, a couple of questions that I have. If you have the (14;16) or (14;20), would you delay getting bortezomib or some kind of proteasome inhibitor or would you just add that to the mix right away?
Dr. Tricot: No. The reality is that patients with (14;16) and (14;20) translocation have the same chance of going into a complete remission as patients who don’t have this translocation. The difference is that in the other patients, the responses last for long periods of time. These patients tend to be short-lived. But you can still debulk and get the patients to very low doses of tumor cells with those drugs. But after you have done that, then you need to come in with these drugs that are specifically targeting the specific translocations. It's not like you would give this to newly diagnosed patients with a trillion myeloma cells. Your first want to debulk with that you know works and when you have only about a million to 10 million cells left, you want to kill those with those specific drugs.
Jenny: Because they might not have to be that strong in order for you to actually target. And I think one of the reasons that one in ten might work that are these compounds that are tested is because I think previously there wasn’t a lot of very gene specific testing being done.
Dr. Tricot: That's true, absolutely true.
Jenny: So you weren’t separating or segmenting and looking through this database to say, okay, we just want to look at the c-MAF or MAFB gene to target that. We're going to look at myeloma in general and we're going to try to screen for thousands of drugs or something that might work. But since myeloma is all different, then how do you know?
Dr. Tricot: Yes, exactly. Again, if you would take this drug ALP and you would give this to a hundred myeloma patients and you would see a response in one or two patients, you would say that's not a very effective drug. But the ones that are responding were all the ones that are the MAFB or c-MAF translocation and you missed that if you didn’t do the gene array of the FISH or whatever.
Jenny: Right. So that's the bonus of the new gene expression or the new genetic testing and the focus on that.
Dr. Tricot: And we think that basing therapy on gene expression profiles is a better way than has been done in the past. But again, we don’t have hard evidence that's the case but we think that that's the case.
Jenny: I guess I would stress to patients that if you -- I went to attend a patient seminar. This was four years ago when I was diagnosed. They asked all the patients to raise their hand if they knew their genetic features. I would say less than 10% of them raised their hand which I thought was crazy because why wouldn’t you want to know what you're dealing with? I just really think it's important for newly diagnosed patients especially to know what they have and that comes with very thorough testing including the genetic testing.
Dr. Tricot: Let me emphasize again that in myeloma patients, there are two things that are important. One is what do their genes show? What are the genetic features? And number two, how extensive is the disease? Those are basically the only two real things that matter in myeloma. How much disease do I have and what are the features of my disease?
Jenny: Because that helps you craft the plan, right?
Dr. Tricot: Exactly, yes.
Jenny: My final question will be just the expected milestones. We already talked a little bit about what you would like to do next, that you like to study it in vitro and then in mouse models. But could you give us an idea of the milestones, how long that might take and the estimated cost in general, a ballpark?
Dr. Zhan: We propose for two year grant. In the first year we can do the myeloma cell lines to test the drug but we need a little more time to collect the primary myeloma samples, so we need the two years for this proposal. Then for this new technology see if we can finish the myeloma cell lines in the first year but for enough clinic samples, we need the two years too. We don’t have a detailed budget but we hope we can apply 300-500K for this proposal.
Jenny: Okay, great. Well, we've been asking that question to everyone just to get an idea of how long the project would take and when it could be seen potentially in the clinic so that's very helpful.
Dr. Tricot: So let me make sure that we all understand that the two aims of this project are number one, the two drugs that we have discussed today work in the mice and in the test tube the way we think they work and do they work in combination. And the second aim is can we find other important genes in MAF patients that we can also use as targets so that we can even do better than with the two drugs we have. So those are the two aims.
Jenny: Okay, perfect. Gary actually had a follow-up question that was a write-in question that he sent. He said, "Was gene deletion 17 seeming to be the largest genetic high risk feature? Is there the same kind of analysis that you've gone through using this genetic database that can be conducted for this deletion as well?"
Dr. Tricot: The 17 is a more difficult thing to do because in contrast to MAFB and c-MAF and MMSET, those translocations are present in 100% of the cells. The deletion 17 is often present in a fraction of the cells which can go from 14% to 100%. So if you try to collect your myeloma cells, you have no idea how many will have the deletion, how many will not have the deletion. And the gene expression profile becomes much more difficult to do because it's often an acquired abnormality.
Dr. Zhan: We published a paper in 2008 in Blood. We identified a signaling pathway for the p53 deletion. In this 14;16 and 14;20 translocation that Dr. Tricot mentioned, the signal or downstream targets are not so unique in the p53 samples.
Jenny: Great. Well, Doctors Zhan and Tricot, we are so happy to have you on the show. You've described something that is so specific that patients with these translocations have got to be just jumping up and down, that people are looking at this for them on their behalf. I think it's remarkable that we could target something so specifically and really help people that are struggling the most, I would say, really find something that could completely change the course of their future.
Dr. Tricot: That's what we are hoping for and I hope we can deliver. But we will certainly try as hard as we can.
Dr. Zhan: That's right.
Jenny: Well, thank you very much for joining us today. We just really appreciate your participation in this project.
Dr. Zhan: Thank you all.
Dr. Tricot: Thank you. Thank you for listening to the Myeloma Crowd Radio Show and the new MCRI series. We believe patients can help support the discovery of a cure and we encourage you all to become involved.
about the author
Myeloma survivor, patient advocate, wife, mom of 6. Believer that patients can help accelerate a cure by weighing in and participating in clinical research. Founder of Myeloma Crowd by HealthTree and the HealthTree Foundation.
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