The primary cilium is a microtubule-based organelle that extends from the cell surface. It serves as a sensory hub, detecting external stimuli and orchestrating key signaling pathways, like the Hedgehog (Hh) pathway. It has been extensively studied due to its critical role in embryonic development and tissue homeostasis. Its dysregulation can contribute to the development of various cancers. Additionally, the primary cilium structure and function are highly regulated and defects can lead to a broad range of disorders known as ciliopathies.
As aberrant Hedgehog pathway is a driving force in tumor development, phenotypic screening campaigns have been conducted successfully, with discovery of various small molecule inhibitors. Not only their usage as anti-cancer drugs is relevant, they serve as essential tools to uncover signaling biology. However, the identification of the cellular target and mechanism-of-action (MoA) of compound hits is often challenging, vastly reducing their potential. CRISPR-based screens have also shown great potential to identify novel modulators of the Hh pathway. However, many screen hits lack validation and mechanistic insights into their involvement in the signaling cascade remain unresolved. Additionally, as much as discovery of novel pathway modulators is important, the exact role of known key components of the pathway is yet to be fully characterized.
The first project centers on the development of a CRISPR-based screening approach to decipher the MoA of novel Hh pathway inhibitors. After successfully conducting the CRISPR-KO screen, we identified and validated resistance mechanisms against both upstream and downstream Hh pathway inhibitors, through KO of Mosmo and Paxip1, respectively. We used the screening strategy to investigate a novel Hh pathway inhibitor, hit 7, demonstrating its similarity to SMO inhibitors and its effect on the protein itself. As treatment resistance remains a major obstacle in cancer therapy, our findings provide new insight to develop preventive strategies to bypass such issue. We expect this screening strategy to become an important complement to existing methodologies for MoA studies of novel inhibitors.
In the second project, we utilized the CRISPR-KO screening approach to confirm the importance of previously discovered, but unvalidated, gene hits. We focused on characterizing the regulatory function of NF2 on different steps of Hedgehog pathway transduction. We demonstrated that NF2 not only is required for Hedgehog pathway responsiveness to stimulation but is also important for proper function and maintenance of the primary cilium. Identifying these novel pathway components is fundamental as it expands our understanding of both the Hedgehog pathway and the organelle.
Finally, we developed tools to study the ciliary membrane tension, by combining the HaloFlipper and ciliary expression of the HaloTag. We successfully monitored membrane tension differences in the inner leaflet of the primary cilium upon pathway activation. Apart from further dissecting PTCH1 inhibitory function in the Hh pathway, the role of ciliary membrane tension remains unexplored, yet it could be a key factor in the organelle mechano-sensory function.
In conclusion, the work described herein advances several genetic and chemical technologies to enhance our understanding of ciliary signaling - and inhibition thereof - at a molecular level.
