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Mount Sinai, Dharmacon Target Prostate Cancer With siRNAs

Name: John Martignetti
Position: associate professor, Mount Sinai School of Medicine
Background: Assistant professor, Mount Sinai School of Medicine — 1998-2006
Fellow, human genetics, Mount Sinai Hospital — 1996-1997
Resident, pediatrics, Mount Sinai Hospital — 1994-1995
Intern, pediatrics, Mount Sinai Hospital — 1993-1994
MD/PhD, medicine/neurobiology, Mount Sinai School of Medicine — 1993
MPhil, molecular biology, Cambridge University — 1987
BA, biochemistry, Columbia College — 1984

Last month, the Prostate Cancer Foundation announced it had awarded $6.1 million in funding to 63 research investigators to support research projects “with the greatest potential to improve survival and reduce side effects and death for men with advanced prostate cancer.”
One of those grant recipients was John Martignetti, a researcher at the Mount Sinai School of Medicine who is exploring the therapeutic potential of siRNAs — designed with help from Dharmacon — targeting the novel gene KLF6-SV1.
Last week, RNAi News spoke with Martignetti about his research and his one-year, $100,000 grant from the PCF.
Before we jump into the award and the RNAi work, could you give an overview of your lab?
Primarily the lab is a gene-discovery-based one looking for the genes that underlie a number of disorders. Historically, [we’ve] really focused on a lot of Mendelian traits — we’ve identified genes for a number of disorders in bone dysplasia, oncocytopenias, and some hereditary cancer syndromes. More recently, using all the tools and knowledge we’ve built for [those efforts], we’ve started looking at more complex traits … in particular in cancer. 
One of the genes that we originally identified with [Mount Sinai researchers] Scott Friedman … and Goutham Narla … was this KLF6, or Kruppel-like factor 6, gene. Indeed, the first [data] we published together on this was in a 2001 Science paper, which identified KLF6 as a prostate cancer tumor-suppressor gene.
This is where [graduate student and collaborator] Analisa DiFeo came in … [helping us in] trying to understand the biology of KLF6: What are the cancers that it is involved in, what is the method of inactivation, and then what’s the biology associated with it? The hope was that if we could find some insight into one of those roads, we might be able to think about diagnostics and therapeutics.
Over the years what we’ve seen is this gene is actually inactivated in a large number of different cancers including ovarian cancer, colorectal cancer, hepatocellular cancer, glioblastoma, head and neck cancer — a large number of major human cancers. What’s become really interesting is one of the chief mechanisms of inactivation, and it turns out to be, at least from our studies, alternative splicing.
About two or three years ago we published a paper with groups from [Johns] Hopkins, [the] Mayo [Clinic], and [the Fred Hutchinson Cancer Research Center] looking at 3,400 men with prostate cancer. What we did was an association study looking for potential SNPs that might be inherited in the germline in KLF6 that might be associated with an increased risk of prostate cancer.
From that study, we identified a SNP that increased lifetime prostate cancer risk of men with and without a family history [of the disease] by about 50 or 60 percent. … The intriguing part of it was that the SNP was intronic — it was just upstream of exon 2. We then spent a long time trying to understand why that would be, and Ana and Goutham really were the drivers in showing that that SNP resulted in changes in the alternative splicing of that gene, which we had not even realized was going on. So that four-exon gene essentially made four variants — it made the wild-type gene and three isoforms.
One of [those isoforms] in particular, which we’ve named KLF6-SV1 for splice variant 1, is really the focus of this prostate cancer grant that we got and a lot of the work we’re driving forward now.
What is really interesting is this SV1 is actually antagonistic to the tumor suppressor function of KLF6; it actually seems to be growth-promoting. So within the gene itself, it turns out that it actually encodes its antagonist and may have a pro-proliferative form as opposed to just the tumor suppressor. It’s really a nice paradigm in cancer genetics where one gene can be both a tumor suppressor and an oncogene.
Analisa, Goutham, and I have been … trying to understand this variant. It is actually over-expressed in a number of cancers, [including] late-stage cancers. We’ve already published in prostate cancer and ovarian cancer, and have papers coming out now in head and neck cancer, in glioblastoma, and in hepatocellular cancer where this variant is over-expressed, and associated with late-stage disease and possibly even survival.
So it becomes a great target for siRNA, and that’s what we’ve been focusing on. We’ve been using the siRNA to try regulating the gene itself and see if we can probe into the biology. Ana has already shown it is involved in angiogenesis [and] in cell-cell communication. Again, we’re probing along [into] all the things that make a tumor cell more aggressive where we see this gene being over-expressed … and Ana has been using siRNA to [do that] in many different cancer lines.
Dharmacon has been a partner … in trying to help us [design those siRNAs] because, as you can imagine, with an isoform you’ve got to be pretty careful about how you pick the sequences so you only affect your isoform and not any of the others. They’ve also been helpful in giving us second- and third-generation molecules that are much more stable in vivo.
With the prostate cancer grant and [related] ovarian [cancer] studies … Ana is … targeting the SV1 in vivo using animals and bioluminescence to show the effect of SV1 inhibition on tumor formation, growth, and the overall survival of animals, in particular models.
What’s the status of the work right now?
All of the [earlier] work was done primarily in cell culture. … Now we’ve moved to [animal models]. … Working with Dharmacon, we’ve set up dosing schedules and amounts [of siRNAs] to see if we can change the tumor growth in vivo.
While the work is applicable to a number of cancers, what are the specific goals for the prostate cancer project?
We’ve devised a couple of models [and in one what we’ll be doing is a] two-pronged [experiment]. We’ll be injecting luciferase-positive prostate [cancer] cells that Ana has engineered directly into the prostate. Using the bioluminescence, we can see how good we’ve been and monitor the growth of these cells. [Then we will] treat [the animals] systemically [with siRNAs] to see if we can change … the local growth and see if that makes a difference in the advancement of these cells to a more metastatic model — will they escape beyond the local environment of the prostate to affect the seminiferous vessels, to affect the lymph nodes, [et cetera]?
The other [project] that Ana has engineered quite nicely with one of the technicians in the lab [involves] a metastases model where we take these [cancer] cells and inject them into the left side of the heart. The reason we do that is to avoid pulmonary circulation. … We can monitor the time it takes metastases to form and how long it takes for them to grow in the animal. [Then, with this model], we can try using systemic siRNA to try to make a change in that pattern, which seems to be very consistent in all the animals.
How long is the award supposed to fund this research?
Only one year. We’re running as fast as we can to try doing these things, but clearly these are going to be long-standing experiments.
As it stands with Dharmacon, do they have rights to compounds? Are they a commercial partner?
Right now, we have a research agreement in place. Mount Sinai … and Dharmacon have worked out the respective ends of what happens with this [work]. All the intellectual [property] on KLF6 and KLF6-SV1 inhibition [is held by] Mount Sinai. For the siRNAs, that’s got to be worked out with Dharmacon.

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