Dr. Arul M. Chinnaiyan Video (Text Version)
Therapeutic Targeting of SPINK1-Positive Prostate Cancer
Natasha Kyprianou
Good morning; my name is Natasha Kyprianou and I welcome everybody to the scientific session this morning. Gene fusions, the androgen receptor, metabolic effects, SNP (snip) analysis, immune checkpoints, microRNAs. Since its inception, the PCRP program has invested in innovative research resulting in the major breakthroughs that have changed the landscape of prostate cancer research. This morning it is my privilege to lead, together with my co-chair Mr. Westley Sholes, the session on Game-Changing Research, where the nation’s top investigators and most innovative minds will present the groundbreaking discoveries in the detection, treatment, genetics, and the epidemiology of prostate cancer. This is where the journey begins; high innovation leading to strong impact in the clinical disease.
It is my honor to introduce the opening speaker for today’s Game-Changing session, Dr. Arul Chinnaiyan. Dr. Chinnaiyan is a Hicks-endowed Professor and Professor of Pathology and Urology at the University of Michigan at Ann Arbor. He’s a Howard Hughes Investigator as well as an American Cancer Society Professor. His seminal discoveries include the recurrent gene fusions of TMPRSS2 to ETS family transcription factors as markers of prostate cancer. He and his team developed the popular cancer profiling bioinformatics resource called Oncomine, which I’m sure most of us—a lot of us -- use it. Dr. Chinnaiyan will present his recent findings on the therapeutic targeting of gene fusions in prostate cancer; Dr. Chinnaiyan.
Arul M. Chinnaiyan, M.D., Ph.D.; Hicks-Endowed Professor and Professor of Pathology and Urology, University of Michigan, Ann Arbor
Thank you Natasha and Wes for the kind invitation and I’d like to begin by thanking the DOD Prostate Cancer Research Program for funding our research for approximately the last decade, really since the beginning of my laboratory. So today I’ll actually be focusing on a recent story that was just published in the Journal of Science Translational Medicine, and it really stems from an Idea award that we received here from the DOD Prostate Cancer Research Program, and it builds upon our initial discoveries of gene fusions in prostate cancer.
So as you heard from Natasha, in 2005 we discovered that a majority of prostate cancers harbor gene fusions or translocations of an androgen or testosterone regulated gene that gets fused upstream of a family of oncogenic transcription factors called the ETS family. This then makes a lot of sense in the context of prostate cancer biology because this then makes these oncogenic transcription factors under the control of androgen and it gives you a potential molecular basis of how anti-androgens may function. Not only do we believe that this is one of the molecular basises for prostate cancer, but since the discovery—before this initial discovery, it was really thought that gene fusions and translocations were primarily relegated to the hematologic malignancies and rare soft tissue tumors. More recent data stemming from this initial study suggests that gene fusions in translocations are a common mechanism in cancer in general.
So this is a pie chart describing the prevalence of these different gene fusions in prostate cancer. In shades of blue are basically the ETS gene fusions that we identified. And they represent over 50% of prostate cancer. TMPRSS2 is the most common; a recent finding of fusions of the RAF pathway both B-RAF and C-RAF which we reported last year in Nature Medicine actually is a discovery of a potentially targetable class of gene fusions and prostate cancer. And what I’ll be primarily focused on today is really this class of prostate cancer, which is about 10% that is an outlier expression of SPINK-1. Each of these classes of prostate cancer are mutually exclusive in that we don’t find foci that have any one of the--that have both of these aberrations.
The other thing just to mention about the gene fusions in general is the way that we’re beginning to translate this is--is that this ends up being an exquisitely specific biomarker for prostate cancer so we’re beginning--potentially this year we’re going to be introducing a test to potentially detect these gene fusions in the urine of men and prostate cancer. Long term of course, we would like to target these gene fusions because we believe that this is essentially the BCR-able equivalent for prostate cancer and that particular disease, that gene fusion represents a CML and there’s a nice therapy for CML. So what we’d like to see in the future is that we could begin to target this particular molecule in the context of prostate cancer. But one of the ongoing challenges is that this is a transcription factor and it’s not a kinase, so this is one of the challenges that we’re going to have to look forward to in the future.
So a little bit of background on SPINK-1, SPINK-1 is a serine protease inhibitor; also known as Pancreatic Secretory Trypsin Inhibitor. It basically functions in the pancreas to block proteolysis and premature activation of different enzymes. It’s been found previously to be elevated, have elevated expression in a number of different cancer types. We initially discovered this in 2008 that about 10% of prostate cancers harbor overexpression, or a pathogenic overexpression we believe, of this molecule SPINK-1 and found it to be mutually exclusive with the ETS fusions in that if you’re positive for SPINK-1, you are negative for the ETS gene fusions and vice versa. So this essentially defined a discreet molecular subtype of prostate cancer that was characterized by not having an ETS-based gene fusion.
In that initial study, we found that this particular marker actually ascribes a set of patients that have a more aggressive form of prostate cancer. And we showed this on more than 1,000 patients across three different data sets, one a microarray data set at University of Michigan immunohistochemistry cohort and about 800-patients from Memorial Sloan Kettering immunohistochemistry cohort that high levels of SPINK-1 are associated with more aggressive prostate cancer than those patients that have a low level of SPINK-1. So that was our initial study.
In 2010 a group from Finland corroborated our studies in their cohort and they found again that SPINK-1 defined an aggressive subtype of prostate cancer. So this is the expression of SPINK so one of the things that we wanted to do was to try to identify preclinical models that we can begin to study SPINK-1 in the context of both—as a biomarker, as well as an a-therapeutic target. And so what we found was that the 22RV1 cell line was a SPINK-1 positive prostate cancer while all of the other cell lines were negative for SPINK-1. VCAP is well known as being the TMPRSS2/ERG-positive prostate cancer as well as H660 and LNCAP as well as MDAPCA2B are basically ETV-1 cryptic insertions of ETV-1.
The other thing to mention and what was attractive about SPINK-1 was that it was secreted so it potentially was an extracellular target that we could go after unlike the ETS gene fusions which were transcription factors and are going to require a considerable amount of development to develop small molecule inhibitors. We were very—we gravitated towards SPINK-1 because it was an extracellular target and you could think about monoclonal antibody-based therapy. What we found was that, when we added SPINK-1 to a variety of prostate cell lines, it induced cell proliferation, so it was essentially functioning as potentially an autocrine or paracrine factor.
Similarly when we again added SPINK-1 to benign prostate epithelial cells, RWPE, this induced cell invasion. Similarly when we took conditioned media from 22RV1 cells that have high expression of SPINK-1 and added that condition media back to these benign prostate epithelial cells, it also induced invasion. When we look at the 22RV1 cells which are SPINK-1 positive and we knock down SPINK-1 expression, we blocked their invasive ability, and we can rescue this effect by adding back recombinant SPINK-1 or adding conditioned media from 22RV1 cells, so again implicating SPINK-1 as a potential oncogenic factor at least in these in vitro assays.
So as I mentioned to you earlier, one of the reasons we were attracted to this was that this was an extracellular target so we evaluated a blocking antibody to SPINK-1 and showed that when we added this blocking antibody to 22RV1 cells we actually could block cell proliferation and this is shown here, the change in curves here. And what we found was that this was in a SPINK-1 specific way in that prostate cancer cell lines that were a positive for SPINK-1—these two are actually derivatives from the same patient, invasion was blocked specifically in these prostate cancer cell lines while other prostate cancer cell lines which are negative for SPINK-1 had no effect by this blocking antibody.
Next we wanted to look into the mechanisms of how SPINK-1 essentially functions and what’s interesting is that SPINK-1 has these immunoglobulin repeat domains and has a similar structure to EGF and in another particular—in another cancer, another group actually identified that SPINK-1 potentially binds to EGFR. So what we found in prostate cancer was that SPINK-1 does indeed bind to EGFR, epidermal growth factor receptor, and this is shown here. Interestingly, when we add SPINK-1 to 22RV1 cells which are positive for SPINK-1; these are again starved cells, we can then add—we can then activate EGFR signaling. This is represented by phosphorylation of EGFR and this was similar to adding EGF directly to 22RV1 but not necessarily as robust.
The other thing that we found was that SPINK-1 was actually mediating a part of its effects through the EGFR receptor in that when we knocked down EGFR receptor in 22RV1 cells we blocked their ability to invade and we could rescue that partially by adding back recombinant SPINK-1. And this then brought up the idea that we could potentially take advantage of an already FDA-approved drug, Cetuximab, which is essentially a blocking antibody against EGFR and what we show here that in SPINK—in a SPINK-1 preferential fashion that the EGFR antibody, Cetuximab was blocking cell invasion in the 22RV1 cells and not having an effect on SPINK-1 negative cell lines.
What we found was that this was a—a partial effect in that a monoclonal antibody to SPINK-1 had a more profound effect than the Cetuximab or the EGFR monoclonal antibody. And we can get a slight additive effect when we combined both antibodies. What was interesting was that unlike the SPINK-1 monoclonal antibody, the Cetuximab antibody did not affect cell proliferation. So there is a difference between the Cetuximab, the EGFR monoclonal antibody and the SPINK-1 monoclonal antibody.
So the next step we did was to take these studies into a preclinical model in vivo to again sort of begin to emulate what we might see in patients. What we found was that in 22RV1 xenografts, we could block their growth in vivo, and this is shown in the red line here relative to an isotype-matched immunoglobulin, and we would get an intermediate effect with a monoclonal antibody against EGFR or Cetuximab. So this is showing that in vivo we could block tumor growth in a xenograft model and when we combined a monoclonal antibody to SPINK-1 with Cetuximab, you actually get an additive effect. And this is shown here. So this is the evidence that in a preclinical model in vivo, we could block SPINK-1 positive tumor growth. And this was again specific to prostate cancers that were positive for SPINK-1. When we look at PC3 xenografts, a monoclonal antibody against SPINK-1 or Cetuximab had no effect. So again, this is in the context of a biomarker-informed context.
So what are the clinical implications of this? Certainly Cetuximab has been tried in clinical trials and this is a trial that Susan Slovin coordinated looking at Cetuximab in prostate cancers and this was relatively—this particular trial was not particularly optimistic because a very small percentage of patients actually responded. About three out of 36 patients had a 50%–80% decline in PSA. But again this particular trial was not done in a biomarker-informed context in that they were essentially treating patients with Cetuximab and Doxorubicin in an all comers’ fashion. So the thought is that these particular patients may actually be SPINK-1 positive, and we’re trying to investigate that hypothesis in collaboration with Susan as well as Howard Scher at Memorial Sloan Kettering.
So just to wrap up, SPINK-1 defines 10% of prostate cancers that are negative for the ETS and RAF-based gene fusions. SPINK-1 is an oncogenic factor in vitro in vivo. SPINK-1 partially mediates its effects through the EGFR receptor. It’s not a complete functional connection there. The SPINK-1 monoclonal antibody, and, to a lesser effect, the EGFR monoclonal antibody, blocks SPINK-1 effects both in vitro as well as in vivo preclinical models. The thought is that Cetuximab or the EGFR monoclonal antibody which is already FDA approved for certain indications could be or should be evaluated in a SPINK-1 biomarker-informed clinical trial. And we present the rationale really for developing a humanized SPINK-1 monoclonal antibody for the evaluation in clinical trials. Of course, we would need to also look into the toxicity of potentially blocking SPINK-1 because there are levels of SPINK-1 in a normal tissue such as a pancreas and colon.
So this is from a review summarizing this from Owen Witte’s group at UCLA, showing that how SPINK-1 is functioning in the context of engaging the EGFR receptor potentially is one mechanism; and the therapeutic targeting of SPINK-1 could involve monoclonal antibodies against EGFR or potentially neutralizing antibodies against SPINK-1.
So this then brings together an ongoing or an overall vision for prostate cancer into the future that we believe that prostate cancer will be able to be molecularly subtyped into different classes. As I mentioned to you earlier, 50%–60% of prostate cancers harbor these ETS gene fusions that we discovered. About 2% harbor RAF-based gene fusions and about 10% harbor SPINK-1 overexpression. Not only will this be potentially important in the context of developing different biomarkers, but we actually believe that this may actually be useful in predicting therapy for different types of prostate cancer. For example, we have data suggesting that ETS gene fusion-based prostate cancers may actually be susceptible for PARP inhibitors, SPINK-1 you might speculate would be susceptible to SPINK-1 monoclonal antibody or EGFR inhibitors and certain the RAF-based gene fusions are candidates for RAF and MEK kinase inhibitors. The problem with the RAF fusions is that they’re a very low percentage of prostate cancer, only about 2% but they’re particularly aggressive. These patients have very high Gleason Grade prostate cancers and generally have metastatic disease.
So with that I’d like to wrap up and this work—much of this work was carried out by Bushra Ateeq who couldn’t be here today. She’s a postdoctoral fellow in the laboratory. I’d like to also thank Scott Tomlins who is in the laboratory as an M.D., Ph.D. student and made the initial discovery of gene fusions in prostate cancer and also made the discovery that SPINK-1 was an outlier gene in ETS negative prostate cancers; I’ll put in a plug for Felix Feng who I believe has a poster here as well as Qi Cao who both are also funded by the Department of Defense. And this project was primarily funded by a DOD Idea Award, as well as various ancillary support from the Prostate Cancer Foundation as well as from the NCI-EDRN and SPORE Programs. Thank you for your attention.