While working in the lab of C. David Allis, PhD, at the Rockefeller University in New York, Dr Phillips set out to uncover the mechanism of a drug called MI-2, which successfully killed DIPG cells both in cultured cell lines and animal models. MI-2 made sense as a candidate, Dr Phillips said, because it works by modifying chromatin structure, and DIPG results from mutations in histones, a key chromatin component.
In leukemia, MI-2 targets a protein called menin, but menin had never been implicated in glioma before. After a variety of experiments in glioma cells led nowhere, though, Dr Phillips began to wonder if menin might not mediate MI-2’s killing in this case. Sure enough, when he knocked out menin from DIPG cells using CRISPR, the cells continued to grow vigorously — but they also remained susceptible to MI-2.
“We went on a bit of an odyssey to figure out what the drug was doing,” Dr Phillips said. “It was still an interesting candidate, because it was still not toxic to normal cells and still pretty good at killing tumor cells.” Eventually, the researchers uncovered the drug’s target in glioma, the cholesterol biosynthesis enzyme lanosterol synthase.
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“We’re not saying that MI-2 doesn’t bind to menin,” Dr Phillips pointed out. “We’re saying that yes, it may bind to menin, but in the context of glioma, that isn’t relevant to its effect.”
The result points to the complexity of identifying effective targeted therapies. “Drugs typically hit multiple targets, and it is important to know which one is relevant in a given system,” said Dr Phillips.
Dr Califano also stressed that point, calling it “the really exciting result” of the paper by Sheltzer’s team. “The repertoire of proteins that are being affected by a drug is much larger than we originally thought, and reproducibly so,” he said. “We need to start thinking of drugs not as ‘one target-one drug’, but [as] ‘one mechanism of action, many targets.’” To pursue this approach, Dr Califano started a company called Darwin Health, which takes a more comprehensive view of drug development.
“We know that tumors have a million ways to escape treatment,” he said. Even if a drug targets a truly essential protein, he said, inherent redundancy allows the cancer to develop a way around it. By looking at the drug’s effect on homeostatic regulation, rather than focusing on individual targets, he said, “it’s like the cancer cell is a building that has 30 columns that keep it up, and now you’ve taken out all 30 [columns], instead of just taking out 1 [column] and hoping the building will come down.”
Multitarget activity, coupled with a misunderstanding of the target’s necessity in the cell, can lead to failure in clinical trials due to toxicity, said Lin. “When it binds to a nonessential protein, it doesn’t kill the cells,” she explained. “As you increase the concentration, you get cell death, but at the same time, now you’re hitting the toxicity and the clinical safety issue.”
She cautioned against sweeping generalizations from the study results, however. “We’re not saying that the genes we chose and the drugs that we tested don’t have therapeutic potential,” she said. “We are testing for specific cancer dependency.” Even though the cells could survive without a particular protein, she said, a drug targeting that protein could still be a useful component of a combination therapy.
Once they showed that none of the 10 drugs targeted the proteins they expected, Lin and her colleagues wondered: what, exactly, do they target? They developed cancer cells resistant to 1 of the drugs, OTS964, thought to target the kinase PBK/TOPK. Sequencing revealed that all the resistant cells had a mutation in the kinase domain of CDK11. Introducing that mutation by CRISPR editing confirmed it was the target of OTS964.
This is the first known inhibitor of CDK11, which could become a promising target for individualized therapy.
References
- Lin A, Giuliano CJ, Sayles NM, Sheltzer J. CRISPR/Cas9 mutagenesis invalidates a putative cancer dependency targeted in on-going clinical trials. eLife 2017;6:e24179. doi: 10.7554/eLife.24179
- Wong CH, Siah KW, Lo AW. Estimation of clinical trial success rates and related parameters. Biostatistics. 2019;20(2):273–286.
- Lin A, Giuliano CJ, Palladino A, et al. Off-target toxicity is a common mechanism of action of cancer drugs undergoing clinical trials. Sci Transl Med. 2019;11(509):eaaw8412.
- Putcha P, Yu J, Rodriguez-Barrueco R, et al. HDAC6 activity is a non-oncogene addiction hub for inflammatory breast cancers. Breast Cancer Res.2015;17(1):149.
- Phillips RE, Yang Y, Smith RC, et al. Target identification reveals lanosterol synthase as a vulnerability in glioma. Proc Natl Acad Sci U S A. 2019;116(16):7957-7962.
