Exploiting a freshly discovered splicing mechanism to target brain cancer
Glioblastoma (GBM) is a devastating disease with very few therapeutic options, partly because high-throughput genetic approaches suffer under the high heterogeneity of GBM. Frequently, targeted agents have yielded poor results in clinical trials due to a lack of predictive biomarkers to stratify patients who could benefit.
Recently, researchers from the renowned David H. Koch Institute of Integrative Cancer Research at MIT published an article in the journal Cancer Cell which could open up new avenues for GBM treatment.
For years, these researchers had been working on a high-throughput screening approach to identify GBM dependencies which they can exploit to gain a deeper understanding of the disease and develop new treatment strategies. In one of their screenings, they discovered PRMT5, an arginine methyltransferase that seemed to slow down cancer growth. Upon validation in multiple in vitro and in vivo systems, the researchers found that PRMT5 inhibition caused the cells to lose S-phase and arrest in in G2/M, furthermore these effects seem to be dependent on the enzymatic function of PRMT5.
So far, so good. But then the problems started.
First, while some GBM cell lines were susceptible to chemical inhibition of PRMT5 with a drug, others were resistant, something often observed due to the high heterogeneity of the underlying disease. Second, even cell lines susceptible to the drug, once put into the brain of mice (intracranial xenograft), the benefits of PRMT5 inhibition were underwhelming, mostly because crossing the blood-brain-barrier (BBB) is very hard pharmacologically. Only mice who had very progressed brain tumors which lost BBB integrity responded to the drug. Third, and worst of all, PRMT5 is known to effect a wide variety of biological processes, making it very hard to predict which patients could ever benefit if they invest time and money to generate next-generation PRMT5 inhibitors able to cross the BBB.
They were stuck.
At every project, there comes a point where one has to make a decision to either drop it or double down. The sunken cost fallacy is a real danger. Big pharma usually has to drop a candidate if the smallest obstacles occur because clinical trials are so expensive, whereas researchers have a bit more leniency.
In this case, Braun CJ et al., decided to double down and discover what exactly is going on with PRMT5. They took an absurd amount of GBM cell lines, treated them with 11 different concentrations of PRMT5 inhibitor, and clustered them into responder and resistant cell lines. Then they ruled out obvious resistant targets like drug-efflux pumps and cross-referenced the cell lines for common mutations, none of which was apparent to explain the resistance. Finally, they compared the whole transcriptomes between resisters and susceptible cell lines and discovered that resisters were enriched in genes for RNA processing and splicing. Among 59 genes, they discovered that the ratio of two candidates, CLNS1A and RIOK1, had the highest predictive value for PRMT5 susceptibility, whereas PRMT5 expression levels were surprisingly less predictive. Notably, those two genes are mutually-exclusive co-factors which direct PRMT5 activity towards snRNP or ribosomal biogenesis.
This finding urged the researchers to look more closely at splicing.
PRMT5 inhibition caused a profound disturbance in alternative splicing, leading to an upregulation of detained introns in mRNA transcripts. Only recently, Boutz P. et al. had discovered that detained introns are not caused by splicing errors, but presented a novel regulatory function of retaining certain mRNA transcripts in the nucleus.
Imagine detained introns as equivalents to the “#” sign in a programing language; for as long as these are included, your code (transcript) is not going to be read by the machine (ribosome).
Transcripts with detained introns are trapped in the nucleus until they eventually decay, consequently a cell can use detained introns to downregulate certain proteins by depleting the message.
But why would cells ever want to do that?
The final piece of the puzzle came together once the researchers analyzed which messages got depleted by PMRT5 inhibition.
It turned out that transcripts containing detained introns were associated with cell cycle, DNA replication and chromosomal segregation; all pathways that are typically downregulated in post-mitotic neurons.
In a last effort to combine everything they have learned, the researchers looked at neuronal differentiation. Here they discovered that neuronal stem cells start out with high levels of PRMT5 (which means low levels of detained introns) and as the cells mature and become full-fledged neurons, PRMT5 levels drop (detained introns in certain mRNAs increase) and the neurons stop proliferating.
Collectively, these data suggest that the splicing of sets of DIs is differentially regulated during neuronal differentiation, thereby cooperating with transcriptional changes to coordinate cell-cycle exit and terminal differentiation. -Braun CJ et al., 2017
While the authors were initially searching for a mechanism to explain why certain cancer cells are sensitive or resistant to PRMT5 inhibition, they discovered a more general principle of how cells evolved diverse mechanisms to regulate molecular programs.
Cancer is evolution on steroids, only once we understand the nuances of how a normal cell works we can hope to grasp why cancers are so diverse in finding ways to survive. Braun CJ et al.’s deep dive into PRMT5 biology ultimately discribes an additional way how this malignant disease can transform post-mitotic neurons to proliferative glioblastoma cells.
We hypothesize that the strong upregulation of PRMT5 mRNA levels in malignant gliomas is phenocopying the high levels of PRMT5 mRNA in stem cells to enable their proliferative capacity.
In summary, Braun CJ et al.’s research provides a solid theoretical basis of developing targeted therapy against a previously unknown subset of malignant cancers; those who abuse the intron detainment mechanism of post-mitotic cells to circumvent non-proliferation.
One plus for therapy is already clear:
Patients losing these intron detainments will be easy to spot.
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