As interest grows in better understanding the highly complex post-transcriptional environment in tumors, RNA-binding proteins have emerged as a class of potential cancer therapeutic targets. Recent research implicates them in MYC-driven cancers and in acute myeloid leukemia, to name just two examples, and the list is likely to lengthen.
RNA-binding proteins (RBP) are an emerging class of potential cancer therapeutic targets, albeit with myriad complexities that have yet to be untangled. Recent research has implicated RBPs in MYC-driven cancers and in acute myeloid leukemia (AML)—two examples in a lengthening list.
“The early genomics era was mainly focused on DNA-level molecular mechanisms—transcription factors and epigenetic modifiers were seen as the drivers of gene expression control,” remarks Hani Goodarzi, PhD, of the University of California, San Francisco. “But some RNA biologists, including Robert Darnell's group at The Rockefeller University [in New York, NY], were already looking ahead to how RBPs might also modulate gene expression.” Notably, Darnell developed cross-linking immunoprecipitation (CLIP) to pinpoint to which RNA type, and where, a given protein binds.
“Once we realized that transcriptional regulation is only one part of the equation, more researchers began paying attention to what happens after, in the post-transcription space,” Goodarzi adds.
Interest in RBPs picked up, and lately “the floodgates have opened,” notes Gene Yeo, PhD, of the University of California, San Diego, in terms of tools at RNA biologists' disposal. These include mass spectrometry–based quantitative proteomics and STAMP, a technology his group developed to study RBP–RNA interactions in single cells (Nature Methods 2021;18:507-19). As well, CRISPR–Cas9 screening “has become very important for looking at synthetic lethality and function to better identify RBPs as novel drug targets.”
Occasionally, toolbox components are tweaked and refined; for instance, Yeo's team came up with “enhanced CLIP”—dubbed eCLIP—which is less technically demanding, with improved specificity and success rates. eCLIP “enabled us to generate large-scale interaction maps of RBPs and their targets,” he says. “It turns out that there are considerably more of these proteins than previously thought; some 10% to 20% of the human genome encodes RBPs” (Nature 2020;583:711-9).
Another novel technique for mapping RBP networks, called HyperTRIBE, was adapted from studying nerve cells in fruit flies by Michael Kharas, PhD, of Memorial Sloan Kettering Cancer Center, also in New York, NY (Nat Commun 2020;11:2026). For Kharas, “the most powerful aspect of RBPs is their ability to really change the cell state, because they influence protein production, dictating how much or how little is made from a given transcript.” Cancer can interfere with this process, he adds, “tipping a delicate balance and thereby altering key decision points for the cell.”
“RBPs are complex molecules whose activities reverberate throughout the cell's gene expression network,” Goodarzi concurs. Whereas transcription factors such as p53 have long received the spotlight as key regulators that tumor cells frequently hijack for their own purposes, “we're learning that RBPs are master regulators, too, and also co-opted” in cancer. As well, “in sequencing more cancer genomes, we're starting to uncover a ton of mutations in RBPs,” he notes, “which has really put them on the map for cancer biologists.”
A recent study from Yeo's group sheds new light on cancers addicted to MYC, which has long vexed researchers as a therapeutic target. However, probing the post-transcriptional milieu of MYC-driven tumor types may yield workaround strategies down the road (Mol Cell 2021;81:3048–64).
Yeo reported that YTHDF2, an RBP, is a vulnerability in triple-negative breast cancer (TNBC) with hyperactivated MYC. YTHDF2 typically keeps a lid on the number of mRNA transcripts that are translated, earmarking many for degradation to maintain cellular homeostasis. With MYC addiction, transcription and translation levels are aberrantly high, so YTHDF2 “becomes more important than ever” for balance, he explains. “When we inhibited it, that provoked a lot of cellular stress from accumulated unfolded proteins, which then triggered apoptosis” in TNBC cells and tumor xenografts.
“Our findings show how cancer cells exploit the function of specific RBPs, to evade stress-induced death,” Yeo adds. “To us, YTHDF2 is a plausible therapeutic candidate, but of course there are others out there.”
“Others” may include RBMX and RBMXL1, which Kharas and his team have been studying. After initially identifying the RBP Musashi-2 as an important regulator in AML, they began scoping out Musashi-2′s network, landing on RBMX and RBMXL1. Both are overexpressed in AML and necessary for tumor cell survival (Nat Cancer 2021;2:741-57).
“We found that these two RBPs directly promote the transcription of their target, CBX5, itself a regulator of chromatin accessibility in AML cells,” Kharas explains. “Knocking them out reduced CBX5′s mRNA and protein abundance, changing how chromatin is compacted, which stunted cell growth and delayed leukemia development.”
The number of identified RBPs is estimated at 1,500, so deciding which ones to pursue therapeutically “will come down to prevalence,” Goodarzi says. “If an RBP's mode of regulation is pretty extensive, impacting a broad set of cellular and cancer states, that opens the door for it to be prioritized.”
For instance, SF3b1—a key RNA splicing component—is frequently mutated in patients with myelodysplastic syndromes, which can morph into leukemia. H3B-8800 (H3 Biomedicine), a small molecule that modulates SF3b1′s activity, is one drug being evaluated in the clinic. However, in preliminary data from a phase I trial of 15 patients there were no objective responses (Leukemia 2021 Jun 25 [Epub ahead of print]).
A challenge is that RBPs “have different functions that are wholly context-dependent; the same protein that's a splicing factor in the nucleus can be a stability factor in the cytoplasm,” Goodarzi says.
Yeo agrees: “You'd need to discern what, exactly, to target—is it an RBP's RNA recognition function, its ability to recruit other proteins as part of a complex, or something else entirely?” Compounding the complexity, many RBPs have intrinsically disordered regions, “which are structurally unstable and can form aggregates. We don't yet know if this aspect would make them easier or harder to drug.”
Strategies being explored include not only small-molecule inhibitors, but antisense oligonucleotides that modulate RBPs at their own transcript level. As well, decoy RNAs conjugated to proteolysis-targeted chimeras (PROTAC) could selectively trap RBPs, routing them toward degradation. This RNA–PROTAC concept has shown utility in vitro, targeting LIN28 and RBFOX1 in cancer cell lines.
“The more we learn about RBPs, and with better technology, the fancier we can get in thinking about how to drug them,” Kharas says. “This is just the beginning.”
“I'd say RNA is having a renaissance moment,” Goodarzi adds. “We understand very little about post-transcriptional control. It's this vast landscape, and we've barely scratched the surface. But the tools keep improving, and excitement in the field is driving participation, so now is a pretty great time to be an RNA biologist.” –Alissa Poh