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The cell cycle (CC) underpins diverse cell processes like cell differentiation, cell expansion, and tumorigenesis but current single-cell (sc) strategies study CC as: coarse phases, rely on transcriptomic signatures, use imaging modalities limited to adherent cells, or lack high-throughput multiplexing. To solve this, we develop an expanded, Mass Cytometry (MC) approach with 48 CC-related molecules that deeply phenotypes the diversity of scCC states. Using Cytometry by Time of Flight, we quantify scCC states across suspension and adherent cell lines, and stimulated primary human T cells. Our approach captures the diversity of scCC states, including atypical CC states beyond canonical definitions. Pharmacologically-induced CC arrest reveals that perturbations exacerbate noncanonical states and induce previously unobserved states. Notably, primary cells escaping CC inhibition demonstrated aberrant CC states compared to untreated cells. Our approach enables deeper phenotyping of CC biology that generalizes to diverse cell systems with simultaneous multiplexing and integration with MC platforms. Subject terms: Assay systems, Proteomics, Cell biology, Immunology, Systems biology

Reactivation of the latent viral reservoirs is crucial for a cure of HIV/AIDS. However, current latency reversing agents are inefficient, and the endogenous factors that have the potential to reactivate HIV in vivo remain poorly understood. To identify natural activators of latent HIV-1, we screened a comprehensive peptide/protein library derived from human hemofiltrate, representing the entire blood peptidome, using J-Lat cell lines harboring transcriptionally silent HIV-1 GFP reporter viruses. Fractions potently reactivating HIV-1 from latency contained human Retinol Binding Protein 4 (RBP4), the carrier of retinol (Vitamin A). We found that retinol-bound holo-RBP4 but not retinol-free apo-RBP4 strongly reactivates HIV-1 in a variety of latently infected T cell lines. Functional analyses indicate that this reactivation involves activation of the canonical NF-κB pathway and is strengthened by JAK/STAT5 and JNK signalling but does not require retinoic acid production. High levels of RBP4 were detected in plasma from both healthy individuals and people living with HIV-1. Physiological concentrations of RBP4 induced significant viral reactivation in latently infected cells from individuals on long-term antiretroviral therapy with undetectable viral loads. As a potent natural HIV-1 latency-reversing agent, RBP4 offers a novel approach to activating the latent reservoirs and bringing us closer to a cure. Subject terms: Preclinical research, Infectious diseases

Cancer drug resistance poses a significant challenge in oncology, often driven by intricate cross-talk among membrane-bound receptors that compromise mono-targeted therapies. We develop a dual membrane receptor degradation strategy leveraging Folate Receptor α (FRα) to address this issue. Folate Receptor α Targeting Chimeras-dual (FolTAC-dual) are engineered degraders designed to selectively and simultaneously degrade distinct receptor pairs: (1) EGFR/HER2 and (2) PD-L1/VISTA. Through modular optimization of modality configurations and geometries, we identify the “string” format as the most effective construct. Mechanistic studies demonstrate an ~85% increase in EGFR-binding affinity compared to the conventional knob-into-hole design, likely contributing to the improved efficiency of dual-target degradation. Proof-of-concept studies reveal that EGFR and HER2 FolTAC-dual effectively counteracts resistance in Trastuzumab/Lapatinib-resistant HER2-positive breast cancer models, while PD-L1 and VISTA FolTAC-dual rejuvenates immune responses in PD-L1 antibody-resistant syngeneic mouse models. These findings establish FolTAC-dual as a promising dual-degradation platform for clinical translation. Subject terms: Cancer immunotherapy, Targeted therapies, Protein design, Drug discovery and development