Stem Cell Screen Points to ALS Disease Target

Therapies for amyotrophic lateral sclerosis (ALS) have proven hard to develop, in part because of inadequate disease models. A new stem cell-based screening method may help. Researchers led by Lee Rubin, Harvard University, Cambridge, Massachusetts, screened for compounds that sustain motor neurons derived from mouse embryonic stem cells (ESCs) and ALS patients. As reported in the April 18 Cell Stem Cell online, the kinase inhibitor kenpaullone topped the list of potential drug candidates. It kept motor neurons alive for weeks longer than expected after trophic support was withdrawn. Kenpaullone could point to a new therapeutic target for ALS, and the screening model provides a better way to seek out future compounds, suggested the authors. “When disease-specific cells are part of the process to validate drugs, you are more likely to obtain better results,” Rubin told ARF. Others pointed out that a big leap still exists between cell models and human disease.

Before heading to clinical trials, most drugs are tested on mouse models, but that does not always guarantee success in people. ALS has seen a string of clinical trial failures. To better approximate disease processes, several groups have recently developed human induced pluripotent stem cell (iPSC) models to generate different cell types for drug screens. Although motor neurons have been tricky to obtain from iPS cells in the past (see ARF related news story), researchers now have more reliable methods (see Makhortova et al., 2011) and have used human-derived motor neurons to test a handful of compounds that might treat ALS (see ARF related news story on Egawa et al., 2012).

First author Yin Yang and colleagues grew motor neurons from the embryonic stem cells of wild-type mice or mice carrying the human superoxide dismutase 1 (SOD1/G93A) mutation. SOD1 variants account for a fifth of familial ALS mutations and its misfolded protein aggregates in the disease. To mimic conditions that might cause neurodegeneration in vivo, the group starved the cells of trophic factors or inhibited the PI3K/Akt pathway, which promotes cell survival. The researchers then tested to see if any of the 5,000 compounds in their chemical library kept the cells alive.

A variety of anti-apoptotic compounds did. One in particular—kenpaullone, an inhibitor of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinases—performed better than the rest. After three days of treatment, three- to fourfold more wild-type and mutant SOD1 motor neurons survived with kenpaullone treatment compared to untreated controls. Kenpaullone also maintained neuronal synapse number, structural morphology, and electrophysiological characteristics. The compounds also suppressed SOD1/G93A protein levels in neuronal cultures and reduced its aggregation and ubiquitination. Kenpaullone was first synthesized in the early 1990s, but has never been clinically tested for any disease, said Rubin. GSK-3 activation has been implicated in neurodegeneration, but no GSK-3 inhibitor has succeeded in clinical trials.

Kenpaullone may do more than just inhibit GSK-3, however, as it boosted motor neuron survival more than did other inhibitors of this enzyme or small RNAs that silence the kinase gene. Yang and colleagues found it reduced phosphorylation of both c-Jun N-terminal kinase (JNK) and c-Jun, which help drive stress-induced neuronal apoptosis. Kenpaullone also blocked activation of their upstream regulators, including MKK4, Tak1, and HPK1/GCK-like kinase (HGK), which is expressed in motor neurons. Together, the data suggest that kenpaullone tones down a cell-death signaling cascade, the authors wrote. They suggested that HGK may be a new drug target for ALS.

How does kenpaullone cut the level of SOD1 and prevent it from accumulating? “My guess is that it does not affect protein aggregation directly,” Rubin told Alzforum. Given that the compound curtails both protein levels and its ubiquitination, it probably affects general accumulation processes, he explained. “By keeping cells alive and healthier, it diminishes whatever processes cause the protein to aggregate.”

Yang and colleagues wondered if kenpaullone would affect human motor neurons the same way. To find out, the researchers made motor neurons from the iPSCs of one healthy person and two ALS patients carrying either a SOD1 or TDP-43 mutation. Kenpaullone enhanced cell survival in all three cell types. The researchers compared the compound to two others that recently failed in Phase 3 clinical trials for ALS—olesoxime (see ARF related news story) and dexpramipexole (see ARF related news story). Dexpramipexole did not enhance motor neuron survival. Olesoxime did, but less than kenpaullone and less consistently across cell lines. “We might have been able to predict the clinical trial failure if we had tried the compounds in this human cell system,” said Rubin.

Rubin’s group plans to test iPSC-derived motor neurons from other ALS patients, including those with sporadic disease and non-SOD1 mutations. He also plans to evaluate the compound in an ALS mouse model, though the kinase inhibitor has to be directly delivered into the spinal cord, as it does not easily get into the brain. Rubin has no plans to optimize kenpaullone by medicinal chemistry, but is open to doing so with interested collaborators. His group will also look into HGK as a drug target, he said.

More broadly, these experiments aim to validate this system as a way to screen for new drugs. Techniques for growing human cells are improving, and it may soon be practical to use them for high-throughput screens, said Rubin. Drugs could then be routinely tested on human cells before going into clinical trials, he said.

This comes at a time when some are questioning the value of mouse models of neurodegenerative disorders. “The use of human-derived neural cells is an important addition to drug discovery to help bridge the preclinical-clinical translational gap,” wrote Rebecca Pruss of the pharmaceutical company Trophos in Marseille, France, in an e-mail (see full comment below). Current animal models come with a high translational risk, she wrote. However, Pruss cautioned that it may be difficult to define how activity in such cell models will translate into treatments, pointing out that olexisome showed no benefit in an 18-month trial despite activity in this model. Pruss would have liked to know what riluzole—the only approved drug for ALS—did in this cell model. Rubin said they did test riluzole and it had no effect.

Richard Smith, Center for Neurologic Study, La Jolla, California, also offered cautions. “It is too early to know whether stem cell technology offers a better way to predict clinical results in trials,” he told Alzforum. “Leaping from any model, be it an animal or embryonic stem cell model, is still quite a leap. Nevertheless, anything that advances the identification of molecules that have therapeutic benefit and prove to be useful in clinical trials is extremely welcome.”

Yang YM, Gupta SK, Kim KJ, Powers BE, Cerqueira A, Wainger BJ, Ngo HD, Rosowski KA, Schein PA, Ackeifi CA, Arvanites AC, Davidow LS, Woolf CJ, Rubin LL. A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell Stem Cell. 2013 June 6.

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