Molecular mechanisms of Ras and Receptor Tyrosine Kinase Convergence on PI3K in cancer

Phosphoinositide 3-kinase alpha (PI3Ka), one of the most frequently mutated proteins in cancer, converts phosphatidylinositol 4,5-biphosphate (PIP2) to PIP3, exerting widespread control over signaling pathways including Ras and Akt. In its inactive state, PI3Ka's catalytic domain, p110, is inhibited by the regulatory domain, p85, with activation involving the relief of p85-mediated inhibition by binding to phosphorylated tyrosines on receptor tyrosine kinases (RTKs) and Ras. The Ras/RTK inputs not only release the inhibition of p110 but also serve to recruit PI3Ka to the membrane and presumably promote activating interactions at the membrane bilayer. However, neither RTK nor Ras-mediated interactions with PI3Ka have been directly structurally characterized, and no structures of PI3Ka in the active state while interacting with the membrane have been reported. Given that common cancer mutations in PI3Ka, such as E542K, E545K, and H1047R, are believed to emulate these natural activation mechanisms, elucidating them holds paramount importance in clinical contexts and drug discovery endeavors. Our proposal aims to bridge this gap by characterizing PI3Ka activation at the membrane using advanced biochemical assays and cryo-EM imaging techniques established in our labs. In our preliminary studies we obtained the first cryo-EM reconstructions of the full-length PI3Ka holoenzyme in complex with Ras and phospho-peptides at the surface of lipid nanodiscs. These preliminary reconstructions reveal novel conformational rearrangements between the p110a and p85 subunits, a series of increasingly more active conformations induced by membrane binding and two novel dimeric states of PI3Ka. In Aim 1, we will use biochemical assays with PI3Ka in the presence of phosphorylated RTK tails and KRas on liposomes to understand their cooperation in PI3Ka activation and aim to obtain first high-resolution structural insights into PI3Ka complex with RTK/Ras in the context of lipid nanodiscs. In Aim 2, we will obtain high resolution structures of our newly discovered PI3Ka dimers in the context of larger membrane surface of liposomes and investigate the functional consequences of such dimers. In Aim 3, we will use a combination of mass spectrometry and signaling assays to interrogate how cooperation between RTKs and Ras as activating PI3Ka inputs is fine-tuned in cells by the specific patterns and extent of RTK phosphorylation, and PI3Ka dimerization, to generate distinct signaling inputs. Across all aims, we will investigate how cancer mutations impinge on the physiological mechanisms of PI3Ka activation unveiled by our structural and functional studies. Our ability to conduct structural studies on the full-length PI3Ka in complex with its activating inputs and membrane attachment is innovative and to our knowledge, unprecedented, and will offer a long- awaited physiologically relevant context for mechanistic investigation of oncogenic PI3Ka mutations. Since these mutations influence therapeutic sensitivity, deciphering the mechanisms by which they dysregulate PI3Ka activation could significantly alter patients' treatments. Project Number: 1R37CA308018-01 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Kliment Verba | Institution: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, SAN FRANCISCO, CA | Award Amount: $636,345 | Activity Code: R37 | Study Section: Macromolecular Structure and Function C Study Section[MSFC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11278232