Spatiotemporal Nanotransformers for Antitumoral Orchestration of Protein Homeostasis

Protein homeostasis represents a fundamental cellular process that maintains the delicate balance between protein synthesis, folding, post-translational modifications, and degradation. Throughout the protein life cyclefrom synthesis to degradationcells employ intrinsic biochemical regulatory mechanisms, including the autophagy-lysosome pathway, the ubiquitin-proteasome system, the unfolded protein response (UPR), and the heat shock response. Tumor cells proficiently remodel their proteostatic landscape to support their survival, proliferation and metastasis. The therapeutic strategy of targeting proteostasis to shift it from a pro-tumorigenic to an anti-tumor state faces significant challenges. A major limitation is the lack of precise molecular switches to controllably regulate processes such as the translation of target mRNAs at the ribosome or the engagement of specific downstream degradation routes. Monotherapeutic targeting of individual nodes within the proteostasis network may lead to drug resistance. Consequently, there is an urgent need to develop more precise targets and spatiotemporally controllable regulatory mechanisms. Such advancements are crucial to enhance tumor selectivity and minimize on-target toxicity to normal tissues, thereby enabling dynamic remodeling of proteostasis networks for cancer therapy. −

Over the years, stimuli-responsive biomaterials have been extensively developed to remodel tumor proteostasis, thereby reprogramming the immunosuppressive tumor microenvironment. For example, ultrasound as a precise non-invasive trigger enables ROS generation deep within tissues, leading to oxidative modification of target proteins causing partial unfolding or complete denaturation, can enhance the delivery of mRNA or proteolysis targeting chimeras (PROTACs) through lysosomal escape and promote immune activation. , Beyond that, engineered CAR-T cells, regulated by heat-sensitive promoters, can regulate therapy-resistant genes via near-infrared (NIR) light or focused ultrasound (FUS) in vivo. − Genome-editing strategies can enable localized CAR expression, promoting T-cell activation within tumors while reducing on-target, off-tumor toxicity. Advancing toward deeper and more clinically relevant interventions, radiotherapy-sensitive prodrugs allow precise spatiotemporal control of protein degradation in tumors, minimizing systemic toxicity. Such prodrugs remain inactive until X-ray exposure cleaves the protecting moieties, − releasing the active drug with synergistic antitumor efficacy both in vitro and in vivo.

A pivotal challenge of orchestrating protein homeostasis for cancer immunotherapy resides in achieving precise and durable degradation of diverse targets, encompassing both membrane and intracellular proteins. , This challenge stems from the limitations of conventional degraders, their lack of spatial control, and inability to engage multi-level regulatory mechanisms. To address this challange, Xu and colleagues have designed a set of Photo Thermal TArgeting Chimeras (PTTACs) by conjugating a intracellular charge transfer photothermal/NIR-II fluorophore with two copies of small-molecule protein binders (BMS-1 for PD-L1 and JQ-1 for BRD4 protein) via oligo­(ethylene glycol) spacers (Figure a). PD-L1 is a key immune checkpoint membrane protein frequently overexpressed in tumor cells, BRD4 is an intracellular epigenetic reader that activates IFN-γ-inducible PD-L1 expression. For tumor-specific delivery, these PTTACs were co-assembled with poly­ethylene glycol–poly­(dialkyl­amino­ethyl meth­acrylate) diblock copolymers (PEPA or PDPA) into nanoscale micelles that enabled tumor-specific delivery in 4T1 breast cancer bearing mice. Within acidic tumor environments, PTTACs are released from the nanomicelle structure and self-assemble into 2-D nanosheets (Figure b). The site of releaseat the surface of tumor cells in case of PEPA (pKa 6.8) micelles, inside tumor cells in case of PDPA (pKa 6.3) micellesis based on protonation of tertiary amines (EPA or DPA) which is known to be ultrasensitive pH-tunable by alkyl substituents. Consequently, PTTACs can bind their corresponding target proteins PD-L1 at the cell surface and BRD4 intracellularly with multivalent affinity. Highly localized hyperthermia upon 808 nm laser irradiation denatures and degrades the targeted proteins through autophagy (Figure b). Experimental validation confirmed that the PTTAC platform enabled simultaneous degradation of membrane PD-L1 and cytoplasmic BRD4, leading to enhanced T-cell activation and potent antitumor immunity in the 4T1 breast cancer model.

1.

PTTAC nanoparticle platform for spatiotemporal degradation of membrane protein PD-L1 and cytosolic protein BRD4. (a) PTTACs are designed by conjugating a photothermal NIR-II fluorophore with corresponding small-molecule protein binders (BMS-1 or JQ-1). BMS-1 PTTACs are formulated into PEPA and JQ-1 PTTACs into PDPA nanoparticle micelles, respectively, for intravenous delivery into the tumor. (b) Within the tumor, PTTAC nanoparticle formulations respond to either extracellular acidic (pH_e < 6.8; in case of PEPA) or intracellular acidic (pH_i < 6.0; in case of PDPA) tumor environment by releasing their PTTAC payloads. Subsequent self-assembly of PTTACs into 2-D nanosheets enables the simultaneous targeting of both cell surface protein PD-L1 and intracellular protein BRD4, leading to their autophagic degradation after NIR irradiation. (Created by Dr. Y. Dang from East China Normal University, China.)

This work represents a novel strategy for multi-level protein homeostasis regulation through photothermal targeting chimeras. The design of PTTACs is innovative in several aspects. Notably, the integration of photothermal agents with protein-targeting ligands allows for precise thermal denaturation of specific proteins only upon NIR irradiation, minimizing off-target effects. Interestingly, the self-assembly into 2-D nanosheets enhances both photothermal conversion and binding avidity, enabling efficient protein degradation at mild temperatures. Importantly, the use of pH-activatable nanoparticles enables hierarchical targeting-from tumor tissue to subcellular compartmentsensuring spatially and temporally controlled degradation. Moreover, the autophagy-mediated degradation mechanism bypasses the limitations of conventional ubiquitin-proteasome systems and expands the scope of degradable targets. This approach not only directly ablates immune checkpoint proteins but is also able to transcriptionally suppress adaptive immune resistance via degradation of BRD4, offering a dual-pathway strategy to reverse immunosuppression. In summary, this work provides a robust and generalizable paradigm in which precision protein degradation enables the spatiotemporally-controlled rewiring of tumor protein homeostasis for enhanced immunotherapy.

CRediT: Bruno G. De Geest: Conceptualization, Writing-review & editing. Ernst Wagner: Conceptualization, Writing-review & editing.

The authors declare no competing financial interest.