In Alzheimer’s research, finding a way to safely lower Aβ levels in the human brain remains a central goal. Thus, scientists were excited by a 2010 Nature paper in which researchers led by Nobel laureate Paul Greengard at Rockefeller University in New York City reported that the cancer drug imatinib, trade name Gleevec, suppressed Aβ42 production through a selective mechanism involving a protein they dubbed γ-secretase activating protein (GSAP) (see Sep 2010 news story). The finding suggested a way to target Aβ while sparing other substrates. Many labs began work in this area; however, in the last three years none have published replication of the key findings on GSAP, and some groups have since abandoned the work. A handful of papers presented mixed results that suggested the story may be more complicated than it first appeared.
Now a December 10, 2013, Alzheimer’s and Dementia paper openly challenges the value of the original findings for human therapy development. Researchers led by Bob Olsson and senior author Kaj Blennow at the University of Gothenburg, Sweden, found no effect of imatinib on blood Aβ42 levels in human cancer patients or on Aβ production in several cell-culture models. When asked about the discrepancies, Greengard suggested that Olsson and colleagues may have been using different experimental conditions, and said he and his colleagues continue to investigate the therapeutic potential of GSAP and imatinib.
Henrik Zetterberg at the University of Gothenburg, a co-author with Olsson and Blennow, wrote to Alzforum, “We believe there are many researchers who have tried different aspects of imatinib as an anti-amyloid agent but failed to get supportive data. Those negative results are difficult to publish.”
Failure to replicate major findings has plagued biomedical science, including Alzheimer’s disease research. Some research groups have reported that attempts to replicate published data fail more often than not (see Prinz et al., 2011). The journal Nature recently issued new guidelines to make methods more transparent in hopes of improving reproducibility (see May 2013 news story), and Nature Biotechnology called for more efforts to replicate published research (see its editorial). The Swedish researchers were unable to persuade Nature to publish their data contradicting the original paper.
Does Cancer Drug Show Promise for Alzheimer’s?
Gamma-secretase has long represented a central target for AD therapy development because this enzyme finishes the sequential proteolysis of amyloid precursor protein (APP) to release the Aβ peptide. However, γ-secretase cuts many substrates, and side effects of direct inhibition have scuttled clinical trials (see Aug 2010 news story; Apr 2011 conference story; Aug 2011 conference story). Greengard and first author Bill Netzer previously reported that imatinib inhibited γ-secretase cleavage of APP without affecting other critical substrates such as Notch (see Oct 2003 news story). Exploring the mechanism, the authors reported that imatinib bound the 16 kDa protein GSAP, which is itself the cleavage product of a larger protein. In in-vitro and cellular model systems, GSAP interacted with a subunit of γ-secretase and the β-C-terminal fragment (β-CTF) of APP to facilitate cleavage of that fragment, the authors reported in 2010. In addition, knockdown of GSAP was reported to lower Aβ levels and plaque load in a mouse model of AD, suggesting this mechanism could be targeted therapeutically in people.
Olsson and colleagues wanted to find out if the imatinib results would translate to humans. Besides validating mouse findings on a potential AD treatment strategy, this would confirm the importance of GSAP as a target. To do this, they examined plasma samples from 51 patients who took imatinib to treat chronic myeloid leukemia. If the drug worked as published, the scientists reasoned, Aβ levels in the blood should drop. However, they saw no decrease over 12 months of treatment. To more directly test imatinib’s effects, Olsson and colleagues then applied the drug to three types of cell culture: human embryonic kidney cells that overexpress APP, induced cortical neurons made from people with Down’s syndrome who produce excess Aβ due to having three copies of APP, and mouse primary neurons. In every case, imatinib had no effect on Aβ, even at doses up to 10 μM. The results suggest that imatinib does not reliably lower Aβ in people, casting doubt on the usefulness of pursuing this pathway therapeutically, Olsson said.
Other researchers lauded the work. “This is a cleverly designed human study. The data look very convincing,” Todd Golde at the University of Florida, Gainesville, wrote to Alzforum. He noted that kinase inhibitors such as imatinib can have distinct effects in different cell types. “Under some circumstances, imatinib may have some modulatory effects on Aβ. However, that is not going to be sufficient to warrant clinical testing,” he suggested.
The study adds to an already contradictory literature on imatinib. Some papers have reported that imatinib treatment can lower Aβ, but some details conflict with Netzer and Greengard’s findings. For example, researchers led by Ellen Kilger at the University of Tübingen, Germany, saw a drop in secreted Aβ after administering imatinib to neuroglioma cells, but they traced the mechanism to enhanced degradation of Aβ, rather than inhibition of γ-secretase (see Eisele et al., 2007). A German group using 10 μM imatinib as a control in a study on a different topic recently reported that the drug lowered Aβ in cultured induced human neurons (see Mertens et al., 2013). Researchers led by Dirk Beher at Asceneuron SA, an Alzheimer’s-oriented spinoff of Merck Serono in Geneva, reported that imatinib lowered Aβ levels in cell culture, but did not affect plasma Aβ when administered to rats (see Hussain et al., 2013). “The new paper nicely extends our imatinib findings [from rat to human]. Together these studies put some question marks on the mechanism of action of imatinib, and whether associated targets are worth pursuing for Alzheimer’s disease,” Beher told Alzforum.
Netzer said that differences in experimental technique could explain the discrepancies between his results and those of Olsson et al. He noted that in Olsson’s study the plasma concentrations of imatinib in the cancer patients averaged 1.5 μM, far lower than the 5 μM needed to see an effect on Aβ in his cell culture studies. Imatinib is not very potent at lowering Aβ, Netzer noted. Drugs that are developed for chronic use generally act in the nanomolar range. In addition, imatinib poorly penetrates the brain, so if plasma Aβ originates from the brain, as some studies suggest (see Demattos et al., 2002), peripheral administration of the drug would not be expected to affect levels, Netzer told Alzforum. “In our hands, imatinib is so reliable that we use it as a positive control for lowering Aβ,” he said. He said he has not tested the drug in the cell cultures used by Olsson and colleagues, but has seen it lower Aβ in rat primary neurons and several cell lines. Netzer thinks that compounds related to imatinib, but more potent and brain-penetrant, will be promising therapeutics for AD.
Some researchers interviewed for this article pointed to a 2005 paper from Michael Wolfe and colleagues at Brigham and Women’s Hospital, Boston, for a possible explanation of the discrepancies. Wolfe and colleagues analyzed imatinib preparations by high-performance liquid chromatography, and traced the γ-secretase inhibitory activity Netzer and Greengard had reported in 2003 to a contaminant present in some preparations (see Fraering et al., 2005). This contaminant might represent a breakdown product of imatinib, Wolfe speculated, but he added that since 2005 he has been unable to identify the active compound.
GSAP’s Mysteries Deepen
Reports on GSAP similarly conflict. While some researchers have found an effect on Aβ, no one has confirmed cleavage of GSAP or replicated a direct interaction between it and the other players: APP, γ-secretase, and imatinib. Beher and colleagues found that knockdown of GSAP in cell culture lowered Aβ, but overexpression had no effect. Beher saw no interaction between GSAP and the C-terminal fragment of APP in co-immunoprecipitation studies, nor any effect of GSAP on Aβ in in-vitro γ-secretase assays. “When we looked at direct effects of GSAP on γ-secretase, everything was negative,” Beher told Alzforum. He suggested that GSAP may affect Aβ production or secretion through some indirect mechanism. He is not pursuing the research further.
Likewise, researchers led by Chuck Sanders at Vanderbilt University, Nashville, Tennessee, expressed recombinant GSAP in bacteria, and found that the purified protein did not bind either imatinib or APP β-CTF (see Deatherage et al., 2012). “It’s not the definitive word on GSAP, but it was negative enough that we think the story must be more complicated than we thought originally,” Sanders told Alzforum. He has also stopped work on the protein. He noted that although the published papers show varied data, “In all cases, the results are not what you would have expected based on the original work.”
In addition to Hussain et al. and Deatherage et al., Lawrence Rajendran at the University of Zurich, whose lab recently published RNAi-based screens on APP processing (see Udayar et al., 2013; Bali et al., 2012), told Alzforum that he has been unable to replicate the key findings but declined to comment on the details as his paper is still under consideration at Nature. Rajendran believes there may be an alternate explanation for GSAP’s effect on Aβ.
Other researchers declined to be identified but privately told Alzforum they could not reproduce the results and had abandoned this line of research. In total, eight γ-secretase research groups confirmed that they were unable to replicate aspects of the data. Olsson said he tried to collaborate with other labs, but all the groups he contacted told him they had canceled research in this area. Golde wrote to Alzforum, “As far as I know, there has been no high-quality independent replication of the GSAP data.”
Greengard suggested that the problem may arise from the chemical characteristics of GSAP. The protein is extremely hydrophobic and aggregates easily, he told Alzforum. In his own lab, researchers see inconsistent results in in-vitro assays based on whether GSAP has aggregated or not, whereas knockdown of the protein in cells always lowers Aβ, he said. He considers the key findings valid, and is currently looking for other proteins that bind GSAP and might make attractive therapeutic targets.
The Challenge of Reproducibility
How to make sense of the conflicting reports? Gerhard Multhaup at McGill University, Montreal, noted that small differences in experimental techniques can greatly affect results. The current papers compare apples to oranges, he told Alzforum. In order to refute a study, “You have to try to reproduce the data by using the exact same methods,” he said. For GSAP and imatinib, this has not yet been done, he added.
Pharmaceutical company researchers Alzforum spoke with about this issue tended to disagree that a failure to replicate is valid only if the exact procedures were repeated with the exact same models and reagents. They maintained that in order for a finding to be of practical use in a therapy development setting, it has to be robust enough that it can be replicated in several different models and with slight variations in technique and reagents.
Given the difficulties with reproducing GSAP and imatinib data, some researchers wondered why Nature has not published a Brief Communication Arising on the original paper, a preferred format for follow-up data. When asked that, Olsson told Alzforum he indeed had submitted his paper first to Nature as a BCA, but that the journal rejected it after soliciting comments from Greengard and two independent reviewers. According to Blennow, the original authors argued in their review that the Swedish paper must be wrong because it was unable to replicate the original cell and animal results in patients. "We see it the other way round," Blennow said. The AD field abounds with examples of therapies that looked promising in mice but failed in clinical trials.
A spokeswoman from Nature declined to discuss how this paper was handled, citing journal policy of not commenting on individual manuscripts. She noted that Nature’s policy is to send BCAs first to the lab that produced the original paper. The BCA and the original authors’ response are then sent to independent referees. “The decision to publish a BCA is based solely on whether the independent referees believe that the BCA presents a conclusive challenge to an aspect of the original paper,” the spokeswoman wrote to Alzforum. She added that the process can be customized and decisions are evaluated on a case-by-case basis. The same editor who handled the original paper generally handles the BCA.
Scientists frequently bellyache about the peer review process, but usually in private. It is rare that complaints around publication of follow-up data or replication attempts spill into the public. In 2010, this happened for a different Aβ paper, when a follow-up study prompted a comment by Adriano Aguzzi.
“It is crucial for the sake of science that deviating opinions are heard, and preferably at the same level as the original research,” Bart De Strooper at VIB and KU Leuven, Belgium, wrote to Alzforum. He serves on the editorial board of EMBO Molecular Medicine and eLife. “We should, as a community, accept debate, and discussion should prevail over dogma. The journal Science did a wonderful job when publishing the opposing views on the bexarotene paper,” he added (see May 2013 news story). He believes the GSAP story should be handled similarly. “Nobody has been able to confirm the GSAP data. Had negative data been published earlier, it would have saved a lot of time, money, and expectations,” he said.—Madolyn Bowman Rogers
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