Discovery and Clinical Proof-of-Concept of RLY-2608, a First-in-Class Mutant-Selective Allosteric PI3Kα Inhibitor That Decouples Antitumor Activity from Hyperinsulinemia

PIK3CA (PI3Kα) is a lipid kinase commonly mutated in cancer, including ∼40% of hormone receptor–positive breast cancer. The most frequently observed mutants occur in the kinase and helical domains. Orthosteric PI3Kα inhibitors suffer from poor selectivity leading to undesirable side effects, most prominently hyperglycemia due to inhibition of wild-type (WT) PI3Kα. Here, we used molecular dynamics simulations and cryo-electron microscopy to identify an allosteric network that provides an explanation for how mutations favor PI3Kα activation. A DNA-encoded library screen leveraging electron microscopy-optimized constructs, differential enrichment, and an orthosteric-blocking compound led to the identification of RLY-2608, a first-in-class allosteric mutant-selective inhibitor of PI3Kα. RLY-2608 inhibited tumor growth in PIK3CA-mutant xenograft models with minimal impact on insulin, a marker of dysregulated glucose homeostasis. RLY-2608 elicited objective tumor responses in two patients diagnosed with advanced hormone receptor–positive breast cancer with kinase or helical domain PIK3CA mutations, with no observed WT PI3Kα-related toxicities. Significance: Treatments for PIK3CA-mutant cancers are limited by toxicities associated with the inhibition of WT PI3Kα. Molecular dynamics, cryo-electron microscopy, and DNA-encoded libraries were used to develop RLY-2608, a first-in-class inhibitor that demonstrates mutant selectivity in patients. This marks the advance of clinical mutant-selective inhibition that overcomes limitations of orthosteric PI3Kα inhibitors

Mise en évidence de la fonction pro-apoptotique des récepteurs Unc5h (implications dans le développement du système nerveux et la tumorigenèse)

Les récepteurs à dépendance sont des protéines qui activent des fonctions très diverses en présence de leur ligand (croissance axonales, migration cellulaire, différenciation...) et induisent l'apoptose si celui-ci n'est plus présent. Une cellule exprimant un tel récepteur est ainsi dépendante de la présence de son ligand pour sa survie. Je me suis intéressé au cours de ma thèse à un groupe particulier de ces récepteurs, les récepteurs Unc5h. Ces derniers ont pour ligand la nétrine-1, une protéine impliquée dans le guidage des axones en croissance et des neurones en migration. Les travaux présentés dans cette thèse ont permis de montrer que les récepteurs Unc5h induisent l'apoptose en absence de nétrine-1 et sont donc des récepteurs à dépendance. Le mécanisme de cette activité pro-apoptotique semble nécessiter le clivage des récepteurs Unc5h et dépend des caspases, une famille de protéases centrales de la signalisation apoptotique. De plus, ce mécanisme semble contrôlé par la nétrine-1 via l'induction de l'oligomérisation de ces récepteurs. Ces travaux ont aussi abouti à la mise en évidence d'un partenaire pro-apoptotique d'un de ces récepteurs, La DAP-kinase (Death-Associated-Protein-Kinase), une protéine impliquée dans différentes voies de signalisation apoptotiques. La découverte de cette double fonction Guidage/Apoptose pourrait permettre d'éclairer le rôle des récepteurs Unc5h dans le développement du système nerveux et la tumorigenèseLYON1-BU.Sciences (692662101) / SudocSudocFranceF

The dependence receptor Trkc triggers mitochondria-dependent apoptosis upon Cobra-1 recruitment

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor’s intracellular domain, leading to the formation of a proapoptotic “killer” fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1

The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase

Netrin-1 receptors UNC5H (UNC5H1–4) were originally proposed to mediate the chemorepulsive activity of netrin-1 during axonal guidance processes. However, UNC5H receptors were more recently described as dependence receptors and, as such, able to trigger apoptosis in the absence of netrin-1. They were also proposed as putative tumor suppressors. Here, we show that UNC5H2 physically interacts with the serine/threonine kinase death-associated protein kinase (DAP-kinase) both in cell culture and in embryonic mouse brains. This interaction occurs in part through the respective death domains of UNC5H2 and DAP-kinase. Moreover, part of UNC5H2 proapoptotic activity occurs through this interaction because UNC5H2-induced cell death is partly impaired in the presence of dominant-negative mutants of DAP-kinase or in DAP-kinase mutant murine embryonic fibroblast cells. In the absence of netrin-1, UNC5H2 reduces DAP-kinase autophosphorylation on Ser308 and increases the catalytic activity of the kinase while netrin-1 blocks UNC5H2-dependent DAP-kinase activation. Thus, the pair netrin-1/UNC5H2 may regulate cell fate by controlling the proapoptotic kinase activity of DAP-kinase