The programmed death ligand 1 interactome demonstrates bidirectional signaling coordinating immune suppression and cancer progression in head and neck squamous cell carcinoma.

BACKGROUND: The programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) are validated cancer targets; however, emerging mechanisms and impact of PD-L1 intracellular signaling on cancer behavior are poorly understood. METHODS: We investigated the cancer cell intrinsic role of PD-L1 in multiple patient-derived models in vitro and in vivo. PD-L1 overexpression, knockdown, and PD-L1 intracellular domain (PD-L1-ICD) deletion (Δ260-290PD-L1) models were assessed for key cancer properties: clonogenicity, motility, invasion, and immune evasion. To determine how PD-L1 transduces signals intracellularly, we used the BioID2 platform to identify the PD-L1 intracellular interactome. Both human papillomavirus-positive and negative patient-derived xenografts were implanted in NOD-scid-gamma and humanized mouse models to investigate the effects of recombinant PD-1, anti-PD-L1, and anti-signal transducer and activator of transcription 3 (STAT3) in vivo. RESULTS: PD-L1 intracellular signaling increased clonogenicity, motility, and invasiveness in multiple head and neck squamous cell carcinoma (HNSCC) models, and PD-1 binding enhanced these effects. Protein proximity labeling revealed the PD-L1 interactome, distinct for unbound and bound PD-1, which initiated cancer cell-intrinsic signaling. PD-L1 binding partners interleukin enhancer binding factors 2 and 3 (ILF2-ILF3) transduced their effect through STAT3. Δ260-290PD-L1 disrupted signaling and reversed pro-growth properties. In humanized HNSCC in vivo models bearing T-cells, PD-1 binding triggered PD-L1 signaling, and dual PD-L1 and STAT3 inhibition were required to achieve tumor control. CONCLUSIONS: Upon PD-1 binding, the PD-L1 extracellular and intracellular domains exert a synchronized effect to promote immune evasion by inhibiting T-cell function while simultaneously enhancing cancer cell-invasive properties.

The Hec1/Ndc80 tail domain is required for force generation at kinetochores, but is dispensable for kinetochore-microtubule attachment formation and Ska complex recruitment.

The conserved kinetochore-associated NDC80 complex (composed of Hec1/Ndc80, Nuf2, Spc24, and Spc25) has well-documented roles in mitosis including 1) connecting mitotic chromosomes to spindle microtubules to establish force-transducing kinetochore-microtubule attachments and 2) regulating the binding strength between kinetochores and microtubules such that correct attachments are stabilized and erroneous attachments are released. Although the NDC80 complex plays a central role in forming and regulating attachments to microtubules, additional factors support these processes as well, including the spindle and kinetochore-associated (Ska) complex. Multiple lines of evidence suggest that Ska complexes strengthen attachments by increasing the ability of NDC80 complexes to bind microtubules, especially to depolymerizing microtubule plus ends, but how this is accomplished remains unclear. Using cell-based and in vitro assays, we demonstrate that the Hec1 tail domain is dispensable for Ska complex recruitment to kinetochores and for generation of kinetochore-microtubule attachments in human cells. We further demonstrate that Hec1 tail phosphorylation regulates kinetochore-microtubule attachment stability independently of the Ska complex. Finally, we map the location of the Ska complex in cells to a region near the coiled-coil domain of the NDC80 complex and demonstrate that this region is required for Ska complex recruitment to the NDC80 complex--microtubule interface.