During an episode of your favorite true crime drama, you’ve probably seen the detective piece together clues with all evidence pointing neatly to “Suspect A”. But there’s something that doesn’t quite add up. Then, during the last ten minutes of the episode comes the plot twist. A new piece of evidence is unveiled, and all signs now point to the previously unsuspected, “Suspect B”. Sometimes, scientific discoveries are like that too, only arguably more exciting since the suspense of such a discovery may build over years, or even decades and result in an important scientific finding. A recent story published in Science Advances from the lab of Dr. Bruce Clurman, a member of the divisions of Human Biology and Clinical Research at Fred Hutch, played out a bit like a crime drama. In this study led by Dr. Markus Welcker, researchers uncovered a phosphorylation site thought to stabilize one of the most important oncoproteins, Myc, actually promotes its degradation and a second previously uncharacterized site similarly signals Myc degradation.
The transcription factor Myc is regarded as one of the most important oncogenes as “most cancers rely on its proliferative effects,” Dr. Clurman explains. Myc’s oncogenic potential correlates with its protein abundance, therefore, if therapies were able to inhibit its activity, this could halt further tumor growth. However, Myc, like most transcription factors, is largely considered “undruggable”. Efforts to target Myc therefore focus on how the protein is stabilized and degraded. These processes are regulated by Myc binding to Fbw7, a protein that brings Myc in close proximity to ubiquitin ligases that tag Myc for proteasomal degradation. The Clurman Lab and others previously identified Myc threonine 58 (T58) as part of a degron, a sequence that signals for protein degradation, which gets phosphorylated and recognized by Fbw7. Curiously, early models proposed that a second nearby site, serine 62 (S62), is phosphorylated by oncogenic signaling to stabilize Myc, but that this site was also required to prime the phosphorylation of the nearby destabilizing T58 site. This model was paradoxical and differed from how phosphorylation regulates Fbw7 binding to other substrates. In other words, something didn’t add up.
Dr. Welcker wrestled with accepting this long-standing model for years until he decided to thoroughly and meticulously dissect this model to see for himself whether the different phosphorylation sites promoted Myc stability or degradation. The authors made a specific mutation at the Myc “destabilizing” T58 site and found that Fbw7 was still able to bind to Myc. However, this interaction was lost with a Fbw7 mutant with impaired ability to bind phosphorylated degrons. This unexpected result strongly suggested that there might be a second degron in Myc that directs Fbw7 binding. Consistent with this, others had suspected that there might be a second degron, threonine 244 (T244), near a cancer-associated mutation hotspot, although this had never been directly tested. The authors then asked whether mutating T58 and T244, alone or together, affected the ability of Fbw7 to bind to Myc. When Myc was mutated at either degron individually, Fbw7 was able to bind to it, however, abolishing both sites prevented binding, suggesting that T244 also regulates Fbw7 binding and Myc stability.
In collaboration with the Zheng laboratory (University of Washington), the Clurman lab meticulously analyzed the importance and function of both degrons using a combination of site-specific mutants, binding and competition assays and crystal structures. In their experiments, the authors also analyzed the corresponding secondary phosphorylation sites of each degron, S62 (thought to be a stabilizing site) and T248. Contrary to current dogma, the authors found that phosphorylation of S62 does not actually stabilize Myc by preventing Fbw7 binding to Myc, but rather, it enhances Myc binding to Fbw7 and promotes its degradation. Additionally, the previously uncharacterized diphosphorylated T244/T248 degron further enhances Myc binding to Fbw7, acting in parallel to T58/S62 phosphorylation. Collectively, the cooperative binding of both diphosphorylated Myc degrons to Fbw7 dimers regulates Myc protein levels. Dr. Welcker explains that one of the key aspects to uncovering the molecular intricacies of Myc-Fbw7 interactions that had puzzled researchers for years lay in ensuring that “both proteins were expressed at physiologically relevant levels.”
Moving forward the Clurman lab aims to understand the cooperativity between the two degrons and identify the signaling pathways involved in phosphorylating the second degron to better grasp what pathways promote Myc degradation. Understanding the specific details about how these proteins interact has important implications for developing Myc-targeted cancer therapies. For years, the field has thought that S62 was a stabilizing site, thus this site has been pharmacologically targeted to de-stabilize Myc and quench its activity in cancers. In light of the recent work in the Clurman lab, the researchers predict that inhibiting the phosphorylation of S62 would actually have the exact opposite effect and promote Myc’s stabilization and activity. This work not only corrects this long-standing model of Myc protein stability but also identifies an additional degron that can be used as a target for Myc-directed therapies. Dr. Clurman comments that these discoveries “open the door for new therapies and changes the view on how to approach them.” Case closed.
This research was supported by the National Institutes of Health and the National Cancer Institute.
UW/Fred Hutch Cancer Consortium member Dr. Bruce Clurman contributed to this work.
Welcker M, Wang B, Rusnac DV, Hussaini Y, Swanger J, Zheng N, Clurman BE. Two diphosphorylated degrons control c-Myc degradation by the Fbw7 tumor suppressor. Sci Adv. 2022 Jan 28;8(4):eabl7872. doi: 10.1126/sciadv.abl7872. Epub 2022 Jan 28. PMID: 35089787; PMCID: PMC8797792.