Hyeong-Reh Choi Kim

Protein aggregation is increasingly recognized as a fundamental feature of cancer biology, particularly in tumors experiencing chronic proteotoxic stress. TP53 is one of the most frequently mutated genes in human cancers, and many cancer-associated missense mutations promote p53 misfolding and amyloid-like aggregation. These aggregates impair wild-type tumor-suppressive functions while conferring oncogenic gain-of-function (GOF) activities that promote tumor progression and therapeutic resistance. Despite decades of effort, direct pharmacological targeting of mutant p53 has remained challenging because of its structural properties, including the lack of a readily druggable pocket, as well as its extensive mutational heterogeneity. Here, we employed the AUTOTAC platform to target mutant p53 aggregates by exploiting aggregation-associated conformational features characteristic of β -sheet-rich oligomers and protofibrillar aggregates. ATC-37 was designed by linking a methylene blue-derived amyloid-binding ligand, which preferentially recognizes β -sheet-rich protein aggregates, to an autophagy-targeting ligand (ATL) that allosterically activates the autophagy receptor p62/SQSTM1. This design enables engagement of amyloid-like mutant p53 aggregates and promotes their recruitment to autophagosomes. ATC-37 exhibits a dual, concentration-dependent mechanism of action. At low concentrations, it promotes autophagic degradation of mutant p53 aggregates and suppresses downstream GOF signaling. At higher concentrations, it induces p62-dependent sequestration of mutant p53 aggregates that exceeds autophagic capacity, leading to proteotoxic cell death. In a PANC-1 pancreatic cancer xenograft model, ATC-37 significantly inhibited tumor growth, reduced amyloid-like mutant p53 aggregates, and attenuated mutant p53-associated GOF programs. Importantly, ATC-37 also enhanced the response of mutant p53 pancreatic cancer cells to radiation, suggesting potential utility in combination with radiotherapy. These findings establish aggregation-state-directed AUTOTAC therapy as a conceptually innovative, mutation-agnostic strategy for targeting aggregation-prone mutant p53 and potentially other aggregation-prone oncoproteins.

Supplementary legends Supplementary Tables 1-3 Supplementary Figures 1-8

Genetic alterations play a pivotal role in various human diseases, particularly cancer. The androgen receptor (AR) is a crucial transcription factor driving prostate cancer progression across all stages. Current AR-targeting therapies utilize competitive AR antagonists or pathway suppressors. However, therapy resistance often emerges due to AR mutations and AR splice variants, such as AR-v7. To overcome this, we developed ATC-324, an AR degrader using the innovative protein degradation technology platform AUTOphagy-TArgeting Chimera (AUTOTAC). ATC-324 was designed to comprise enzalutamide, an AR inhibitor, as a target-binding ligand and YT 6-2, a ligand of the autophagy receptor p62/SQSTM1, as an autophagy-targeting ligand. ATC-324 induces the formation of the AR/p62 complex, leading to autophagy–lysosomal degradation of AR. Importantly, ATC-324 effectively degrades AR mutants frequently detected in prostate cancer and codegrades AR-v7 as a heterodimer with full-length AR. ATC-324 reduces nuclear AR levels and downregulates the target gene expression of AR and AR-v7, leading to cytotoxicity in AR-positive prostate cancer cells. We also provide evidence of the therapeutic potential of ATC-324 in vivo as well as ex vivo bone organ culture. Moreover, ATC-324 remains potent in enzalutamide-resistant prostate cancer cells. These results demonstrate the potential of the AUTOTAC platform to target previously considered undruggable proteins and overcome certain drug resistance mechanisms.Significance:The characterization of an AUTOTAC-based degrader capable of inducing autophagic degradation of wild-type and mutated androgen receptors demonstrates the potential of this approach for targeting castration-resistant prostate cancer and overcoming drug resistance.

Supplementary legends Supplementary Tables 1-3 Supplementary Figures 1-8

Genetic alterations play a pivotal role in various human diseases, particularly cancer. The androgen receptor (AR) is a crucial transcription factor driving prostate cancer progression across all stages. Current AR-targeting therapies utilize competitive AR antagonists or pathway suppressors. However, therapy resistance often emerges due to AR mutations and AR splice variants, such as AR-v7. To overcome this, we developed ATC-324, an AR degrader using the innovative protein degradation technology platform AUTOphagy-TArgeting Chimera (AUTOTAC). ATC-324 was designed to comprise enzalutamide, an AR inhibitor, as a target-binding ligand and YT 6-2, a ligand of the autophagy receptor p62/SQSTM1, as an autophagy-targeting ligand. ATC-324 induces the formation of the AR/p62 complex, leading to autophagy–lysosomal degradation of AR. Importantly, ATC-324 effectively degrades AR mutants frequently detected in prostate cancer and codegrades AR-v7 as a heterodimer with full-length AR. ATC-324 reduces nuclear AR levels and downregulates the target gene expression of AR and AR-v7, leading to cytotoxicity in AR-positive prostate cancer cells. We also provide evidence of the therapeutic potential of ATC-324 in vivo as well as ex vivo bone organ culture. Moreover, ATC-324 remains potent in enzalutamide-resistant prostate cancer cells. These results demonstrate the potential of the AUTOTAC platform to target previously considered undruggable proteins and overcome certain drug resistance mechanisms. Significance: The characterization of an AUTOTAC-based degrader capable of inducing autophagic degradation of wild-type and mutated androgen receptors demonstrates the potential of this approach for targeting castration-resistant prostate cancer and overcoming drug resistance.

The vaccinia virus expression system is known for the efficient production of recombinant proteins with “appropriate” posttranslational modification using desired mammalian cell lines. However, being a replication competent virus, vaccinia virus poses a health threat to immunocompromised individuals and requires biosafety level 2 (BSL2) laboratory precautions, thereby restricting its use by the scientific community. Development of the host range restricted modified vaccinia Ankara (MVA) system has allowed researchers to work with a safer virus even at BSL1. Here, we report on the use of an improved second generation MVA viral system incorporating two selective markers and fluorescent proteins for easier recombinant virus identification. Notably, we demonstrate that this novel system is capable of producing secreted recombinant proteins, a finding not previously reported. Through purification and characterization of wild type and mutant platelet‐derived growth factor D (PDGF D) dimer species, we demonstrate this system is capable of producing the latent full‐length PDGF D dimer, partially processed intermediate dimer (hemidimer), as well as fully processed growth factor domain dimer that show chemical integrity and biological activity. Importantly, this system is amenable to scaling up for the mass production of recombinant PDGF D (rPDGF D) dimer species.

Cell survival depends on dynamic interactions among the signaling pathways that control the endoplasmic reticulum (ER) stress response, macroautophagy/autophagy and apoptotic cell death. Our recent study reported an association of cytoplasmic SQSTM1/p62-mediated autophagy with disease progression and therapy resistance in head and neck squamous cell carcinoma (HNSCC). Synthetic small molecule ligands of SQSTM1 activate autophagic flux by binding the SQSTM1 ZZ domain and promoting self-oligomerization. Importantly, we found that the combination of pharmacological activation of SQSTM1 and therapeutic radiation promotes formation of ubiquitinated CASP8 (caspase 8) aggresomes that lead to apoptotic cell death of HNSCC. This finding suggests the potential for the development of a novel therapeutic strategy involving pharmacological activation of SQSTM1 in intrinsically apoptosis-resistant and therapy-resistant cancer cells.

The autophagy–lysosome pathway and apoptosis constitute vital determinants of cell fate and engage in a complex interplay in both physiological and pathological conditions. Central to this interplay is the archetypal autophagic cargo adaptor p62/SQSTM1/Sequestosome-1 which mediates both cell survival and endoplasmic reticulum stress-induced apoptosis via aggregation of ubiquitinated caspase-8. Here, we investigated the role of p62-mediated apoptosis in head and neck squamous cell carcinoma (HNSCC), which can be divided into two groups based on human papillomavirus (HPV) infection status. We show that increased autophagic flux and defective apoptosis are associated with radioresistance in HPV(-) HNSCC, whereas HPV(+) HNSCC fail to induce autophagic flux and readily undergo apoptotic cell death upon radiation treatments. The degree of radioresistance and tumor progression of HPV(-) HNSCC respectively correlated with autophagic activity and cytosolic levels of p62. Pharmacological activation of the p62-ZZ domain using small molecule ligands sensitized radioresistant HPV(-) HNSCC cells to ionizing radiation by facilitating p62 self-polymerization and sequestration of cargoes leading to apoptosis. The self-polymerizing activity of p62 was identified as the essential mechanism by which ubiquitinated caspase-8 is sequestered into aggresome-like structures, without which irradiation fails to induce apoptosis in HNSCC. Our results suggest that harnessing p62-dependent sequestration of ubiquitinated caspase-8 provides a novel therapeutic avenue in patients with radioresistant tumors.

A hallmark of malignant solid tumor is extracellular acidification coupled with metabolic switch to aerobic glycolysis. Using the human MCF10A progression model of breast cancer, we show that glycolytic switch and extracellular acidosis in aggressive cancer cells correlate with increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), known to induce intracellular signal transduction through the interaction with its cell surface receptor CD63, independent of its metalloproteinase inhibitory function. We found that, in aggressive breast carcinoma, the TIMP-1–CD63 signaling axis induced a metabolic switch by upregulating the rate of aerobic glycolysis, lowering mitochondrial respiration, preventing intracellular acidification, and inducing extracellular acidosis. Carbonic anhydrase IX (CAIX), a regulator of cellular pH through the hydration of metabolically released pericellular CO₂, was identified as a downstream mediator of the TIMP-1–CD63 signaling axis responsible for extracellular acidosis. Consistently with our previous study, the TIMP-1–CD63 signaling promoted survival of breast cancer cells. Interestingly, breast carcinoma cell survival was drastically reduced upon shRNA-mediated knockdown of CAIX expression, demonstrating the significance of CAIX-regulated pH in the TIMP-1–CD63-mediated cancer cell survival. Taken together, the present study demonstrates the functional significance of TIMP-1–CD63–CAXI signaling axis in the regulation of tumor metabolism, extracellular acidosis, and survival of breast carcinoma. We propose that this axis may serve as a novel therapeutic target.