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Q&A: Mount Sinai's John Martignetti on Targeting a Novel Cancer Gene with RNAi

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Name:
John Martignetti

Position:
Associate professor, genetics/genomic sciences, pediatrics, oncology, 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
•MD/PhD, medicine/neurobiology, Mount Sinai School of Medicine — 1993
•MPhil, molecular biology, Cambridge University — 1987
•BA, biochemistry, Columbia College — 1984

Following up on earlier studies, including one funded by a one-year, $100,000 grant from the Prostate Cancer Foundation (see RNAi News, 4/5/2007), John Martignetti and colleagues at Mount Sinai School of Medicine earlier this year published data showing that treatment with an siRNA directed against a novel oncology target, an alternatively spliced isoform of the Kruppel-like tumor suppressor KLF6, could increase survival in a mouse model of ovarian cancer.

This week, RNAi News spoke with Martignetti about his latest work and plans going forward.

Let's start with the target itself. Can you give some background on KLF6-SV1?

Originally, Scott Friedman, Goutham Narla, and I [at Mount Sinai] had identified that the tumor-suppressor gene KLF6 was inactivated in a number of cancers, the first demonstration was in prostate cancer. What we then went on to do was an association study to see if there were any germline changes in men with prostate cancer that could predict an increased risk for development of the cancer.

Working with samples from 3,400 men, we identified a [single nucleotide polymorphism] that doubled the risk of prostate cancer. Intriguingly, the SNP was intronic, and it took us about a year to realize that that SNP enhanced expression of an alternative splice form of the tumor suppressor, which we termed KLF6-splice variant 1.

It turns out that the splice variant 1 loses the terminal zinc fingers and nuclear localization but gains its own independent oncogenic activity, in effect antagonizing the growth suppressive properties of KLF6.

Over the years and through different collaborations and studies, we have been analyzing a number of different cancers to understand the role of this alternatively spliced oncogene. The one theme that we keep coming up with is that in all the cancers we've examined — and these range from prostate to hepatocellular to glioblastoma to … ovarian cancer — is that this alternative splice form, which is normally made during development, is up-regulated in essentially all late-stage cancers.

We actually have two papers now, one in [the Journal of Clinical Investigation] and one in Cancer Research, showing that increased expression of this oncogenic form is actually predictive of recurrence and metastatic disease in prostate cancer and in lung cancer.

Given that we've seen the over-expression of KLF6-SV1 in so many cancers and its association with late-stage disease and recurrence, we sought to develop a therapeutic model inhibiting KLF6-SV1 to establish its potential therapeutic value.

Of the different cancers, the one we thought would be a great model is ovarian cancer. First, late-stage ovarian cancer has a terrible prognosis and is a cancer that is in desperate need for some new therapeutic targets. Second, the cancer tends to spread or disseminate primarily in the peritoneum, so it's really, in essence, in an enclosed space. Ultimately, late-stage disease will extend beyond the peritoneum, but it really is a disease not of metastases initially, but of dissemination. A number of chemotherapy protocols provide treatment directly via intraperitoneal injection. Therefore, we thought peritoneal delivery of siRNA would be a perfect use for this technology and potentially provide a paradigm for the field.

And that's the work detailed in the new Cancer Research paper?

Exactly. We started with a well-characterized model for ovarian cancer progression and spread, [but] with a twist: adding a luciferase marker to our ovarian cancer cell lines such that following their injection into the peritoneum we could follow their growth and spread in real time.

For the treatment regimen, we essentially modeled our dosing scheme for siRNA based on the gold-standard treatment for ovarian cancer, which is six doses of cisplatin or carboplatin and [paclitaxel]. So we gave six doses of the siRNA [in two regimens]: one in addition to cisplatin and one as a single agent, both with survival as the end point.

How were the siRNAs delivered?

Working with Dharmacon, now Thermo Fisher Scientific, we had a formulation that we could inject into the peritoneum.

No vehicle?

No. They were just [Dharmcon's] Accell molecules delivered straight into the peritoneum.

Analisa DiFeo, the lead author of the current study, had previously shown that if we treated ovarian cells prior to implantation with siRNAs targeting KFL6, the tumors grew uncontrollably large. But if you treated the cells with the siRNAs against the splice variant, the tumors completely regress and disappear.

That's why we wanted to work with an in vivo ovarian cancer model wherein the tumor was already known to be growing within the peritoneum — to try mimicking the clinical state of the human disease.

What did you find?

The results were two-fold. First, the treatment with the siRNAs actually enhanced the chemotherapeutic regimen such that the tumor size was getting smaller much more rapidly and the animals survived longer even when given relatively low doses of the siRNA. What was the really neat finding was that as a monotherapy … the higher doses of siRNAs tripled median survival and more than doubled overall survival.

A beautiful part of this work, in addition to the treatment, is figuring out part of the mechanism. It turns out that KLF6-SV1 is actually an anti-apoptotic protein and it binds to Noxa, which is a BH3-only protein. When they bind, they are targeted for degradation.

When we had spoken before, you were doing some work with this target in prostate cancer. What kinds of results were you getting there?

[For prostate cancer], the preclinical model is more difficult because of the type of [disease] spread, but in vitro we're seeing exactly the same thing. The cells will apoptose when treated with the siRNAs.

As part of this current paper, Analisa demonstrated that inhibition of KLF6-SV1 in all cell types we tested, including prostate cancer cells, glioblastoma, breast cancer cells, and HCCcells, results in their apoptosis.

Is this particular target, KLF6-SV1, proprietary? Have you filed patent applications on it?

Yes, Mount Sinai has filed patents on KLF6 and KLF6-SV1 as both diagnostic and treatment entities.

So what's the next step to take these findings forward?

The thing is now to try identifying the other types of cancers to which we can apply this knowledge. How can we essentially target the elimination of SV1 in different cancer types? Can inhibition be an adjunct to other current therapies? And then [we want to] develop the necessary preclinical models we need to demonstrate effectiveness. In addition, we continue to explore the mechanistic basis of these findings.

Are you collaborating with anybody on this? Is the Dharmacon arrangement still in place?

While we still have a fantastic relationship with our collaborators at Dharmacon, given that our focus is now on therapeutic development and usage we have begun exploring additional collaborations that can help us advance these findings and move them towards the clinic.

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