BACKGROUND:Mutations in KRAS and BRAF genes are prevalent in colorectal cancer (CRC), which strikingly promote tumorigenesis and lead to poor response to a variety of treatments including immunotherapy by activating the MAPK/ERK pathway. Thus, there is an urgent need to discover effective therapeutic targets and strategies. METHODS:CRISPR-Cas9 lentiviral knockout library was used to screen the suppressors of anti-PD1 immunotherapy. Bioinformatic analysis was used to analyze the correlation between PHF8 expression and immune indicators in CRC. In vitro and in vivo experiments were utilized to determine the effects of PHF8 on the immune indexes and malignant phenotypes of CRC cells. qRT-PCR, western blotting, immunohistochemical (IHC) staining, and chromatin immunoprecipitation (ChIP)-qPCR assays were used to determine the regulatory effects of PHF8 on PD-L1, KRAS, BRAF, and c-Myc and the regulatory effect c-Myc/miR-22-3p signaling axis on PHF8 expression in CRC cells. RESULTS:This study identified histone lysine demethylase PHF8 as a negative regulator for the efficacy of anti-PD1 therapy and found that it was highly expressed in CRCs and strongly associated with poor patient survival. Functional studies showed that PHF8 played an oncogenic role in KRAS- or BRAF-mutant CRC cells, but not in wild-type ones. Mechanistically, PHF8 up-regulated the expression of PD-L1, KRAS, BRAF, and c-Myc by increasing the levels of transcriptional activation marks H3K4me3 and H3K27ac and decreasing the levels of transcriptional repression mark H3K9me2 within their promoter regions, promoting immune escape and tumor progression. Besides, our data also demonstrated that PHF8 was up-regulated by the c-Myc/miR-22-3p signaling axis to form a positive feedback loop. Targeting PHF8 substantially improved the efficacy of anti-PD1 therapy and inhibited the malignant phenotypes of KRAS- or BRAF-mutant CRC cells. CONCLUSION:Our data demonstrate that PHF8 may be an effective therapeutic target for KRAS- or BRAF-mutant CRCs.

Multimodal treatment of cancer is an unstoppable revolution in clinical application. However, designing a platform that integrates therapeutic modalities with different pharmacokinetic characteristics remains a great challenge. Herein, we designed a universal lipid nanoplatform equipping a ROS-cleavable docetaxel prodrug (DTX-L-DTX) and an NF-E2-related factor 2 (NRF2) inhibitor (clobetasol propionate, CP). This simply fabricated nanomedicine enables superior synergistic molecularly targeted/chemo/radio therapy for lung cancer cascade by a transcription factor-driven ROS self-sustainable motion. Chemotherapy is launched via ROS-triggered DTX release. Subsequently, CP inhibits the expression of NRF2 target genes, resulting in efficient targeted therapy, meanwhile inducing sustained ROS generation which in turn facilitates chemotherapy by overcoming ROS consumption during the DTX release process. Finally, the introduction of radiotherapy further amplifies ROS, offering continuous mutual feedback to amplify the ultimate treatment performance. This strategy is conceptually and operationally simple, providing solutions to challenges in clinical cancer treatment and beyond.

Immunotherapy-involved combination cancer therapy heralds an unstoppable revolution in clinical practice. However, designing a streamlined approach to integrate immunotherapy with other therapies remains a formidable challenge. Canagliflozin, a clinical drug for type II diabetes, has gained growing interest in cancer therapy, albeit its precise mechanism remains largely elusive. Here, we delve into the multifaceted antitumor mechanisms of canagliflozin, including co-suppression of MAPK/ERK and PI3K/AKT signaling pathways, induction of PD-L1 degradation via ubiquitination and sensitization to X-ray irradiation via downregulation of DNA damage repair-related molecules, thereby offering opportunities for synergistic cancer treatment. For this purpose, we designed a universal nanoliposome coated with a PD-L1-targeting peptide to efficiently deliver canagliflozin in vivo. This simple fabricated nanodrug exhibited specific tumor-targeting ability and excellent biosafety. In vivo anti-cancer efficacy evaluation revealed synergistic pathway-targeted therapy from canagliflozin and immunotherapy facilitated by both canagliflozin and peptide. Furthermore, the introduction of radiotherapy further augmented the overall treatment performance. This study will revisit the diverse anti-tumor mechanisms of canagliflozin and its potential in cancer therapy, greatly expanding its clinical indication. In addition, the targeted delivery system of canagliflozin developed in this study will achieve multimodal synergistic cancer therapy, providing solutions to challenges in clinical cancer treatment and beyond.

Immune checkpoint blockade (ICB) therapy using anti-programmed cell death 1 (PD1) and cytotoxic T lymphocyte antigen 4 (CTLA4), either individual or in combination, has emerged as a pioneer in clinical immunotherapy for various human cancers. However, immune-related adverse effects (irAEs) remain the significant hindrances to their clinical development. In particular, immune-related myocarditis has emerged as a fatal adverse event with the highest fatality rate. For this purpose, we report herein the first example of uncoupling efficacy and myocarditis toxicity in checkpoint blockade cancer therapy. This efficient, yet highly safe access to ICB therapy was achieved by a simple nanoplatform that masked anti-PD1/CTLA4 antibodies to albumin via a ROS-cleavable linker. An in-depth investigation of the experimental autoimmune myocarditis (EAM) mouse model revealed the avoiding exacerbation of myocarditis of our nanomedicine. Notably, the on-demand release of antibodies at the tumor sites enables the combined checkpoint antibody activity. The present study uncouples the ICB efficacy and toxicity, and highlights the irresistible charm of TME-responsive nanoantibodies in clinical cancer immunotherapy, offering solutions to challenges in the advancement of TME-responsive drug delivery toward clinical application.

Despite the considerable achievements of antibodies targeting PD-1/PD-L1 in cancer immunotherapy, limitations in antitumor immune response and pharmacokinetics hinder their clinical adoption. Small molecules toward PD-L1 degradation signifies an innovative avenue to modulate PD-1/PD-L1 axis. Herein, we unveil a comprehensive engineering involving the development of new PD-L1 degraders based on the berberine (BBR) and palmatine (PMT) bioactive frameworks and explore their translational potential for cancer immunotherapy using a peptide-drug conjugate strategy. Chemical modifications at the O-9 position of PMT dramatically enhance the PD-L1 degradation capacity. Further conjugation of PMT degraders with an anti-PD-L1 peptide featuring disulfide linkers enables efficient GSH-specific prodrug activation, yielding synergistic immunotherapeutic benefits through both external PD-L1 blockade and internal PD-L1 degradation mechanisms. This work elucidates the compelling charm of the discovery and application of PD-L1 degraders, offering solutions to the challenges in advancing cancer immunotherapy in widespread clinics.

Solute carrier family 39 member 10 (SLC39A10) belongs to a subfamily of zinc transporters and plays a key role in B-cell development. Previous studies have reported that its upregulation promotes breast cancer metastasis by enhancing the influx of zinc ions (Zn 2+ ); however, its role in gastric cancer remains totally unclear. Here, we found that SLC39A10 expression was frequently increased in gastric adenocarcinomas and that SLC39A10 upregulation was strongly associated with poor patient outcomes; in addition, we identified SLC39A10 as a direct target of c-Myc. Functional studies showed that ectopic expression of SLC39A10 in gastric cancer cells dramatically enhanced the proliferation, colony formation, invasiveness abilities of these gastric cancer cells and tumorigenic potential in nude mice. Conversely, SLC39A10 knockdown inhibited gastric cancer cell proliferation and colony formation. Mechanistically, SLC39A10 exerted its carcinogenic effects by increasing Zn 2+ availability and subsequently enhancing the enzyme activity of CK2 (casein kinase 2). As a result, the MAPK/ERK and PI3K/AKT pathways, two major downstream effectors of CK2, were activated, while c-Myc, a downstream target of these two pathways, formed a vicious feedback loop with SLC39A10 to drive the malignant progression of gastric cancer. Taken together, our data demonstrate that SLC39A10 is a functional oncogene in gastric cancer and suggest that targeting CK2 is an alternative therapeutic strategy for gastric cancer patients with high SLC39A10 expression.

Prodrug development is of great interest in cancer therapy. From bio-friendly standpoints, traceless prodrug activation would be an ideal approach for cancer treatment owning to the avoidance of byproduct which might induce side effects in living system. Here, we report a fully traceless strategy for cancer imaging and therapy via a metal-free bioorthogonal ligation triggered by nitroreductase (NTR) overexpressed in solid tumors. The reduction of nitro substrates to amines by NTR and further condensation of amines with aldehydes can be seamlessly combined to yield imine-based resveratrol (RSV) with water as the only byproduct. In comparison with RSV, this precursor exhibited not only the same level of anticancer efficiency both in vitro and in vivo under hypoxia, but also a high sensitivity to hypoxia and much lower perturbation towards normal cells, which holds a great potential of theranostic prodrug for cancer therapy.

Small nucleolar RNA SNORD50A and SNORD50B (SNORD50A/B) has been reported to be recurrently deleted and function as a putative tumor suppressor in different types of cancer by binding to and suppressing the activity of the KRAS oncoproteins. Its deletion correlates with poorer patient survival. However, in this study, we surprisingly found that SNORD50A/B loss predicted a better survival in breast cancer patients carrying wild-type p53. Functional studies showed that SNORD50A/B deletion strongly inhibited the proliferation, migration, invasion and tumorigenic potential, and induced cell cycle arrest and apoptosis in p53 wild-type breast cancer cells, while exerted the opposite effects in p53 mutated breast cancer cells. This was also supported by ectopically expressing SNORD50A/B in both p53 wild-type and mutated breast cancer cells. Mechanistically, SNORD50A/B clearly enhances the interaction between E3 ubiquitin ligase TRIM21 and its substrate GMPS by forming a complex among them, thereby promoting GMPS ubiquitination and its subsequent cytoplasmic sequestration. SNORD50A/B deletion in p53 wild-type breast cancer cells will release GMPS and induce the translocation of GMPS into the nucleus, where GMPS can recruit USP7 and form a complex with p53, thereby decreasing p53 ubiquitination, stabilizing p53 proteins, and inhibiting malignant phenotypes of cancer cells. Altogether, the present study first reports that SNORD50A/B plays an oncogenic role in p53 wild-type breast cancers by mediating TRIM21-GMPS interaction.

Background Mannose, a natural hexose existing in daily food, has been demonstrated to preferentially inhibit the progression of tumors with low expression of phosphate mannose isomerase (PMI). However, its function in thyroid cancer still remains elusive. Methods MTT, colony formation and flow cytometry assays were performed to determine the response of thyroid cancer cells to mannose. Meanwhile, mouse models of subcutaneous xenograft and primary papillary thyroid cancer were established to determine in vivo anti-tumor activity of mannose. The underlying mechanism of mannose selectively killing thyroid cancer cells was clarified by a series of molecular and biochemical experiments. Results Our data demonstrated that mannose selectively suppressed the growth of thyroid cancer cells, and found that enzyme activity of PMI rather than its protein expression was negatively associated with the response of thyroid cancer cells to mannose. Besides, our data showed that zinc ion (Zn 2+ ) chelator TPEN clearly increased the response of mannose-insensitive cells to mannose by inhibiting enzyme activity of PMI, while Zn 2+ supplement could effectively reverse this effect. Further studies found that the expression of zinc transport protein ZIP10, which transport Zn 2+ from extracellular area into cells, was negatively related to the response of thyroid cancer cells to mannose. Knocking down ZIP10 in mannose-insensitive cells significantly inhibited in vitro and in vivo growth of these cells by decreasing intracellular Zn 2+ concentration and enzyme activity of PMI. Moreover, ectopic expression of ZIP10 in mannose-sensitive cells decrease their cellular response to mannose. Mechanistically, mannose exerted its anti-tumor effect by inhibiting cellular glycolysis; however, this effect was highly dependent on expression status of ZIP10. Conclusion The present study demonstrate that mannose selectively kills thyroid cancer cells dependent on enzyme activity of PMI rather than its expression, and provide a mechanistic rationale for exploring clinical use of mannose in thyroid cancer therapy.

Hypoxia is inherent character of most solid malignancies, leading to the failure of chemotherapy, radiotherapy and immunotherapy. Atovaquone, an anti-malaria drug, can alleviate tumor hypoxia by inhibiting mitochondrial complex III activity. The present study exploits atovaquone/albumin nanoparticles to improve bioavailability and tumor targeting of atovaquone, enhancing the efficacy of anti-PD-1 therapy by normalizing tumor hypoxia. We prepared atovaquone-loaded human serum albumin (HSA) nanoparticles stabilized by intramolecular disulfide bonds, termed HSA-ATO NPs. The average size and zeta potential of HSA-ATO NPs were measured by particle size analyzer. The morphology of HSA-ATO NPs was characterized by transmission electron microscope (TEM). The bioavailability and safety of HSA-ATO NPs were assessed by animal experiments. Flow cytometry and ELISA assays were used to evaluate tumor immune microenvironment. Our data first verified that atovaquone effectively alleviated tumor hypoxia by inhibiting mitochondrial activity both in vitro and in vivo, and successfully encapsulated atovaquone in vesicle with albumin, forming HSA-ATO NPs of approximately 164 nm in diameter. We then demonstrated that the HSA-ATO NPs possessed excellent bioavailability, tumor targeting and a highly favorable biosafety profile. When combined with anti-PD-1 antibody, we observed that HSA-ATO NPs strongly enhanced the response of mice bearing tumor xenografts to immunotherapy. Mechanistically, HSA-ATO NPs promoted intratumoral CD8+ T cell recruitment by alleviating tumor hypoxia microenvironment, thereby enhancing the efficacy of anti-PD-1 immunotherapy. Our data provide strong evidences showing that HSA-ATO NPs can serve as safe and effective nano-drugs to enhance cancer immunotherapy by alleviating hypoxic tumor microenvironment.

Many high-affinity peptide antagonists of MDM2 and MDMX have been reported as activators of the tumor suppressor protein p53 with therapeutic potential. Unfortunately, peptide activators of p53 generally suffer poor proteolytic stability and low membrane permeability, posing a major pharmacological challenge to anticancer peptide drug development. We previously obtained several potent dodecameric peptide antagonists of MDM2 and MDMX termed PMIs, one of which, TSFAEYWALLSP, bound to MDM2 and MDMX at respective affinities of 0.49 and 2.4 nM. Here we report the development of gold nanoparticles (Np) as a membrane-traversing delivery vehicle to carry PMI for anticancer therapy. Np-PMI was substantially more active in vitro than Nutlin-3 in killing tumor cells bearing wild-type p53, and effectively inhibited tumor growth in metastasis in a mouse homograft mode of melanoma and a patient-derived xenograft model of colon cancer with a favorable safety profile. This clinically viable drug delivery strategy can be applied not only to peptide activators of p53 for cancer therapy, but also to peptide therapeutics in general aimed at targeting intracellular protein-protein interactions for disease intervention.

A major pharmacological barrier to peptide therapeutics is their susceptibility to proteolytic degradation and poor membrane permeability, which, in principle, can be overcome by nanoparticle-based delivery technologies. Proteins, by definition, are nano materials and have been clinically proven as an efficient delivery vehicle for small molecule drugs. Here we describe the design of a protein-based peptide drug carrier derived from the tetramerization domain of the chimeric oncogenic protein Bcr/Abl of chronic myeloid leukemia. A dodecameric peptide inhibitor of the p53-MDM2/MDMX interaction, termed PMI, was grafted to the N-terminal helical region of Bcr/Abl tetramer. To antagonize intracellular MDM2/MDMX for p53 activation, we extended this protein, PMIBcr/Abl, by a C-terminal Arg-repeating hexapeptide to facilitate its cellular uptake. The resultant tetrameric protein PMIBcr/Abl-R6 adopted an alpha-helical conformation in solution and bound to MDM2 at an affinity of 32 nM. PMIBcr/Abl-R6 effectively induced apoptosis of HCT116 p53+/+ cells in vitro in a p53-dependent manner and potently inhibited tumor growth in a nude mouse xenograft model by stabilizing p53 in vivo. Our protein-based delivery strategy thus provides a clinically viable solution to p53-inspired anticancer therapy and is likely applicable to the development of many other peptide therapeutics to target a great variety of intracellular protein-protein interactions responsible for disease initiation and progression.

Clinical translation of therapeutic peptides, particularly those targeting intracellular protein–protein interactions (PPIs), has been hampered by their inefficacious cellular internalization in diseased tissue. Therapeutic peptides engineered into nanostructures with stable spatial architectures and smart disease targeting ability may provide a viable strategy to overcome the pharmaceutical obstacles of peptides. This study describes a strategy to assemble therapeutic peptides into a stable peptide–Au nanohybrid, followed by further self‐assembling into higher‐order nanoclusters with responsiveness to tumor microenvironment. As a proof of concept, an anticancer peptide termed β‐catenin/Bcl9 inhibitors is copolymerized with gold ion and assembled into a cluster of nanohybrids (pCluster). Through a battery of in vitro and in vivo tests, it is demonstrated that pClusters potently inhibit tumor growth and metastasis in several animal models through the impairment of the Wnt/β‐catenin pathway, while maintaining a highly favorable biosafety profile. In addition, it is also found that pClusters synergize with the PD1/PD‐L1 checkpoint blockade immunotherapy. This new strategy of peptide delivery will likely have a broad impact on the development of peptide‐derived therapeutic nanomedicine and reinvigorate efforts to discover peptide drugs that target intracellular PPIs in a great variety of human diseases, including cancer.

The peptide-derived self-assembly platform has attracted increasing attention for its great potential to develop into multitargeting nanomedicines as well as its inherent biocompatibility and biodegradability. However, their clinical application potentials are often compromised by low stability, weak membrane penetrating ability, and limited functions. Herein, inspired by a natural protein from the seeds of Luffa cylindrica, we engineered via epitope grafting and structure design a hybrid peptide-based nanoplatform, termed Lupbin, which is capable of self-assembling into a stable superstructure and concurrently targeting multiple protein-protein interactions (PPIs) located in cytoplasm and nuclei. We showed that Lupbin can efficiently penetrate cell membrane, escape from early endosome-dependent degradation, and subsequently disassemble into free monomers with wide distribution in cytosol and nucleus. Importantly, Lupbin abrogated tumor growth and metastasis through concurrent blockade of the Wnt/β-catenin signaling and reactivation of the p53 signaling, with a highly favorable in vivo biosafety profile. Our strategy expands the application of self-assembled nanomedicines into targeting intercellular PPIs, provides a potential nanoplatform with high stability for multitargeted cancer therapy, and likely reinvigorates the development of peptide-based therapeutics for the treatment of different human diseases including cancer.

Peptide-tuned self-assembly of macromolecular agents (>500 Da) such as therapeutic peptides offers a strategy to improve the properties and biofunctions of degradable nanomaterials, but the tough requirement of macromolecular therapeutics delivery and a lack of understanding of peptide-based self-assembly design present high barriers for their applications. Herein, we developed a new strategy for nanoengineering macromolecular drugs by an elaborate peptide, termed PSP (VVVVVHHRGDC), capable of directly conjugating with cargo to be a PSP-cargo monomer as building block tending to self-assemble into a well-defined nanoshell with tumor-triggered shape and charge switch. As a proof of concept, conjugation PSP to a D-peptide activator of tumor suppressor p53 termed (PMI)-P-D (1492.5 Da) generated hollow spheres similar to 80 m in diameter named PSP-(PMI)-P-D that disintegrated only in the acidic microenvironment of tumor tissues, followed by integrin-mediated cellular uptake of PSP-(PMI)-P-D monomers. Importantly, PSP-based self-assembly successfully endowed the (PMI)-P-D with long circulation time and high cancer-cell-specific intracellular accumulation. PSP-(PMI)-P-D nanoshells potently inhibited tumor growth in vitro and in vivo by the p53 restoration, while maintaining a highly favorable in vivo safety profile. Out of conventional encapsulation and conjugation, our study showcases a clinically viable novel method to nanoengineer macromolecular agents such as peptide for anticancer therapy and provides a hazard-free alternative strategy for the theranostics delivery.

Peptides are a rapidly growing class of therapeutics with many advantages over conventional small molecule drugs. Dextrorotary (D)-peptides, with increased enzymatic stability and prolonged plasma half-life in comparison with natural L-peptides, are considered to have great potential as recognition molecules and therapeutic agents. However, the in vivo efficacy of current therapeutic D-peptides is hindered by their inefficient cellular uptake in diseased tissues. Methods: To overcome physiological and cellular barriers to D-peptides, we designed a gold-based ultra-small nanocarrier coupled with polylysine (PLL) and a receptor-targeted peptide to deliver therapeutic D-peptides. Using a D-peptide p53 activator (DPA) as a proof of concept, we synthesized, functionalized and characterized gold- and DPA-based nanoparticles termed AuNP-DPA. Results: AuNP-DPA were effectively enriched in tumor sites and subsequently internalized by cancer cells, thereby suppressing tumor growth via reactivating p53 signaling. More importantly, through a series of in vivo experiments, AuNP-DPA showed excellent biosafety without the common side effects that hinder p53 therapies in clinic trials. Conclusion: The present study not only sheds light on the development of AuNP-DPA as a novel class of antitumor agents for drugging the p53 pathway in vivo, but also supplies a new strategy to use D-peptides as intracellular PPI inhibitors for cancer-targeted therapy.