Interfering with mitosis is not a new way to treat cancer. More than a quarter century has passed since Randall Johnson, Ph.D., then at the National Cancer Institute, first showed that Taxol (paclitaxel) works against cancer by poisoning mitosis. Paclitaxel, and the even older vinca alkaloids, function by binding to microtubules, hollow structures that form the mitotic spindle, thus halting cell division. (Tumor cells then die by an unknown mechanism.) For decades, the microtubule was the only mitotic target.
But mitosis, the partitioning of a duplicated genome into two daughter cells, is a complex cellular drama with many actors. The microtubule is merely one. In the last few years, new mitotic targets have emerged in cancer, and inhibitors are moving with remarkable speed into the clinic.
Sidetracking Cell Division
One such target is a protein called KSP, for kinesin spindle protein. In 1985, Ronald Vale, Ph.D., at the Marine Biological Laboratory in Woods Hole, Mass., discovered tiny, ATP-dependent motor proteins that ride on microtubules. He named them kinesins. In 1992, Tim Mitchison, Ph.D., of the University of California, San Francisco, showed that the kinesin KSPalso known as Eg5was involved with the mitotic spindle.
In 1999, Mitchison found a small-molecule KSP inhibitor that created radically abnormal cells featuring monopolar spindles, with chromosome fragments spherically arrayed across the cell instead of pulled toward dual centrosomes. Mitosis halted. "The knowledge that [KSP] could be targeted with small molecules really put it on the map, I think, for a lot of drug companies," said Mitchison, who is now at Harvard Medical School in Boston.
Even before Mitchison's paper came out, the South San Francisco, Calif., biotech company Cytokinetics was screening for KSP inhibitors. (Vale cofounded the company in 1998.) In 2002, Cytokinetics' lead compound, ispinesib, became the first such drug to be tried in humans. In theory, specific KSP inhibitors like ispinesib should target only actively dividing cellsincluding tumors. "Evolution built KSP for only one function, and that's to separate out the spindle poles," said Cytokinetics CEO James Sabry, M.D., Ph.D. "[And] there's only one time in the cell's life that it has two spindle poles, and that's in mitosis."
Cell photomicrographs capture the choreography of KSP inhibition. When the cell cannot separate its spindle poles, the characteristic spherical monopolar spindle appears. The cell, unable to properly separate its chromosomes, stops dead in its tracks, thanks to the spindle checkpoint. The cell "then undergoes a mitotic deatheither an apoptotic death, or a death from mitotic catastrophe," said Sabry. The exact mechanism of cell death is unclear, but it resembles that of paclitaxel.
Window of Vulnerability?
KSP inhibitors, in theory, should have fewer side effects than paclitaxel because normal, nondividing cells do not express KSP, whereas microtubules are present everywhere in the body. Paclitaxel, for example, often causes severe neuropathies, because microtubules are crucial for molecular transport within neurons. KSP inhibitors should affect only dividing cells such as bone marrow stem cellsnot neurons. "So an optimistic view is, you could have similar efficacy to Taxol in killing cancer cells, but you could lose some of the side effects," said Mitchison. "You could have a more specific agent." Said Sabry, "We have not seen any nervous side effects with our compounds."
However, the compounds may still have chemotherapy-like side effects. One tantalizing ideacompletely unprovenis that KSP inhibitors could have special killing power against cancer cells, compared with normal cycling cells, because the cancer cells are uniquely vulnerable. "You frequently have rather severe abnormalities in mitosis in the cancer cells that you don't see in [normal] stem cells," said Mitchison. For example, Wilma Lingle, Ph.D., and Jeff Salisbury, Ph.D., of the Mayo Clinic in Rochester, Minn., have shown that centrosome abnormalities are common in breast cancer cells and lead to genomic instability. "It is possible that [KSP] inhibitors or some other new targets in mitosis might be able to take advantage of that and give some selectivity for blocking mitosis in cancer cells with abnormal division, compared to normal stem cells," said Mitchison. "But that's a hope more than a demonstrated fact."
Answers to these questions should come fairly soon. Cytokinetics, together with GlaxoSmithKline and the NCI, is testing ispinesib in nine separate phase II trials, each in a different tumor type. Three phase Ib combination trials are also under way. A second, more potent Cytokinetics KSP inhibitor is in phase I studies. Meanwhile, at the American Association for Cancer Research (AACR) annual meeting in April, Merck reported that it has KSP inhibitors in advanced preclinical development, and at least a half-dozen other drug companies have active programs. (See table, p. 874.) Will these drugs prove better than the taxanes and vincas? "We don't know yet where or whether KSP inhibitors actually, truly will be effective anticancer agents in man," cautioned Sabry. "Whether [tumor] cells are more sensitive to inhibiting a mitotic kinesin, versus a microtubule ... is something that we are still looking to show."
Selection of Mitosis-Targeting Drugs in Development
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Kinase of the Year?
Meanwhile, an even more popular new target in mitosis has emerged: the aurora kinases. Three drug companies are already testing aurora kinase inhibitors in phase I, and by year's end at least two more should be in clinical testing, with others soon to follow. "Aurora may be the kinase of the year because so many drug companies are interested in finding inhibitors to it," James Maller, Ph.D., of the University of Colorado School of Medicine in Denver, said at the AACR meeting.
The original aurora kinase was identified in 1995 in Drosophila mutants, which were defective in spindle pole behavior. (The name comes from aurora borealis, because it's a phenomenon of polar regions.) Human aurora kinase A was first cloned in 1997, by Subrata Sen, Ph.D., at the University of Texas M. D. Anderson Cancer Center in Houston, with aurora B reported the following year by a group at the South San Francisco biotech company Sugen. A handful of studies established aurora A as a probable transforming oncogene that is overexpressed in many tumors.
Aurora kinases A and B are structurally similar but play very different roles in mitosis. Aurora A localizes in mitosis to the spindle poles and to spindle microtubules and is thought to regulate spindle assembly. Aurora B is found first on the centromerethe chromosomespindle attachment pointthen on the central spindle, and finally at the cleavage site between dividing cells. It's crucial for proper chromosome alignment on the spindle, spindle checkpoint function, and cytokinesis (physical cell division).
Given their importance in mitosisand because they are kinases, and thus potentially druggablethe aurora kinases have become popular drug targets. The multiplicity of functions of the aurora kinases in mitosis give a drug "multiple chances to affect the cell and somehow trigger apoptosis," said Matthew Harding, Ph.D., senior director of development pharmacology at Vertex Pharmaceuticals in Cambridge, Mass. "We have multiple bets down with an aurora kinase inhibitor, rather than just targeting cyclin B or something."
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Vertex was the first company to put its aurora kinase inhibitors into clinical studies last December, but the competition is already intense. "It's going to be like the Kentucky Derby, you know, a whole bunch of aurora kinase inhibitors charging around the track," said Don Payan, M.D., chief scientific officer of Rigel Pharmaceuticals, a South San Francisco biotech company set to enter the clinic this year with an aurora kinase inhibitor.
One puzzling aspect of these drugs is that they somehow bypass the spindle checkpoint, causing the cancer cell to go through mitosis but blocking cytokinesis. The result is a bizarre cell that keeps reproducing its DNA, partitioning it into four or eight or 16 nucleiall in a single cell. (This outcome is typical of aurora B inhibition, which for unknown reasons dominates over aurora A inhibition.) "Eventually ... the tumor becomes so overwhelmed with this huge abundance of DNA that, in an incompletely divided cell, the cell has no choice but to die," explained Gary Schwartz, M.D., a medical oncologist at Memorial Sloan-Kettering Cancer Center in New York.
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Vertex's compound showed strong antitumor effects in mouse xenograft experiments published last year in Nature Medicine, along with some reversible bone marrow toxicity, which was expected from a drug that targets dividing cells. In new experiments presented at this year's AACR annual meeting, researchers from Vertex and from the Dana-Farber Cancer Institute in Boston showed that tumor cells with a defective p53 tumor suppressor gene were more likely to undergo multinucleation and apoptosis than p53 wild-type cells. This is important, since in theory normal dividing cells with intact p53 would be much less likely to die when treated than tumor cells. "You don't want normal cells going through endoreduplication, going through mitosisyou want that [DNA repair] checkpoint to be activated," said Harding.
But data from Schwartz's group, also presented at AACR, contradicted these findings. In lab tests, Astra-Zeneca's aurora kinase inhibitor overrode cell cycle checkpoints in tumor cells with wild-type p53 and generated the typical multinucleated cells, which then died. This finding suggests broader antitumor activity but also raises the possibility that normal cells could acquire multiple chromosome copies (polyploidy). If these cells then somehow survive, the resulting aberrant gene expression could lead to secondary cancers. "It's a theoretical possibility," acknowledged Harding. "But normal [polyploid] cells... have all the right biochemistry to activate apoptosis and destroy themselves."
What will happen to real cancer patients treated with these drugs? "I don't think anyone knows what to expect," said Mitchison. "We really don't know the consequences of making cells polyploid. Why do cells die? Would that predispose to cancer downstream? How are cancer cells and normal stem cells going to compare in that response? I think that's rather uncharted territory."
With all the new activity targeting mitosisboth mitotic kinesins and aurora kinasesanswers won't be long in coming. "I'm keeping my fingers crossed," said Mitchison. "At least in academia, poisoning cell division is considered old-fashionedit's all about signaling pathways. But I personally believe there's a lot more we can do with cell division, and do it in a more cancer-selective way."
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