Project description:Phosphoinositide 3-kinase gamma (PI3Kγ) is implicated as a target to repolarize tumor-associated macrophages and promote anti-tumor immune responses in solid cancers. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid, and dendritic lineages. This dependency is characterized by innate inflammatory signaling and elevation of PIK3R5, which encodes a regulatory subunit of PI3Kγ that we find stabilizes the active enzymatic complex when overexpressed. Mechanistically, we identify PAK1 kinase as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency independently of AKT. PI3Kγ inhibition dephosphorylates PAK1, activates a transcriptional network of NFκB-related tumor suppressor genes, and impairs mitochondrial oxidative phosphorylation. We find that treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukemias with activated PIK3R5, either at baseline or by exogenous inflammatory stimulation. Notably, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukemia xenografts with low baseline PIK3R5 expression, as residual leukemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Taken together, our study reveals acute leukemia dependency on a noncanonical PI3Kγ signaling pathway amenable to near-term evaluation in patients using inhibitors already in clinical development.
Project description:Targeted protein degradation via the ubiquitin proteasome has been established in eukaryotes, however no application has been shown in bacteria so far. In the present work, selective protein degradation is shown using hetero bifunctional drugs, bringing substrates in close proximity to the bacterial ClpC1P1P2 protein degradation complex. We performed a TMT based quantitative proteomics analysis to monitor degradation of human BRDT in Mycobacterium smegmatis. Our data shows selective degradation of BRDT when using bacterial PROTACS, indicating the first successful application of targeted protein degradation in bacteria.
Project description:Phagocytosis is an intensely physical process that depends on the mechanical properties of both the phagocytic cell and its chosen target. Here, we employed differentially deformable hydrogel microparticles to examine the role of cargo rigidity in the regulation of phagocytosis by macrophages. Whereas stiff cargos elicited canonical phagocytic cup formation and rapid engulfment, soft cargos induced an architecturally distinct response, characterized by filamentous actin protrusions at the center of the contact site, slower cup advancement, and frequent phagocytic stalling. Using phosphoproteomics, we identified 2 integrins and their downstream effectors as critical mediators of this mechanically regulated phagocytic switch. Indeed, comparison of wild type and 2 integrin deficient macrophages indicated that integrin signaling acts as a mechanical checkpoint by shaping filamentous actin to enable distinct phagocytic engulfment strategies. Collectively, these results illuminate the molecular logic of leukocyte mechanosensing and reveal potential avenues for modulating phagocyte function in immunotherapeutic contexts.
Project description:G protein-coupled receptor (GPCR) kinases (GRKs) selectively phosphorylate agonist bound GPCRs, thereby switching the signal from G protein-dependent to arrestin-dependent pathways, including receptor internalization and downregulation. There are currently no high-resolution details known about how a GRK recognizes an activated receptor, but most functional studies indicate that its unique N-terminus is key to agonist-dependent phosphorylation. Herein, we report the 7.2 Å cryo-electron microscopy (EM) single particle reconstruction of the rhodopsin-rhodopsin kinase (GRK1) complex. The structure reveals a 1:1 assembly with multiple contact points between the GRK1 kinase domain and rhodopsin, the most prominent being the insertion of the GRK1 N-terminal helix directly into the cytoplasmic cleft formed by rhodopsin in its active state. Cross-linking coupled with mass spectrometry, along with functional studies, confirmed the observed interface between the proteins and revealed regions of the complex that are dynamic.