Targeting DAD1 gene with CRISPR-Cas9 system transmucosally delivered by fluorinated polylysine nanoparticles for bladder cancer intravesical gene therapy.

Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro. Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo. Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications.

The m6A Reader YTHDF2 Promotes Bladder Cancer Progression by Suppressing RIG-I-Mediated Immune Response.

N6-Methyladenosine (m6A) is the most prevalent internal modification of mammalian mRNAs. Recent studies have shown that m6A methyltransferases METTL3 and METTL14 play important roles in urothelial bladder carcinoma (BLCA). To provide a more comprehensive understanding of the m6A regulatory landscape in bladder cancer, we investigated the role of YTHDF2, a crucial m6A reader, in BLCA. YTHDF2 was frequently upregulated at both the RNA and protein level in BLCA. Functionally, YTHDF2 promoted the proliferation and tumor growth of BLCA cells in vitro and in vivo, respectively. Integrative RNA sequencing and m6A sequencing analyses identified RIG-I as a downstream target of YTHDF2. Mechanistically, YTHDF2 bound to the coding sequence of DDX58 mRNA, which encodes RIG-I, and mediated its degradation in an m6A-dependent manner. Knockdown of RIG-I inhibited apoptosis and promoted the proliferation of BLCA cells. Depleting RIG-I was also able to reverse the effects of YTHDF2 deficiency. YTHDF2-deficient BLCA cells implanted orthotopically in recipient mice activated an innate immune response and promoted recruitment of CD8+ T lymphocytes into the tumor bed and the urothelium. Moreover, YTHDF2 deficiency enhanced the efficacy of Bacillus Calmette-Guérin immunotherapy treatment. This study reveals that YTHDF2 acts as an oncogene in BLCA. YTHDF2 inhibits RIG-I to facilitate immune evasion, supporting testing YTHDF2 inhibition in combination with immunotherapy. SIGNIFICANCE: YTHDF2 regulates RIG-I-mediated innate immune signaling to support bladder cancer progression, highlighting the functional importance of m6A modifications in bladder cancer and uncovering therapeutic opportunities to improve patient outcomes.

AIM2 inflammasome activation benefits the therapeutic effect of BCG in bladder carcinoma.

A large proportion of bladder cancer (BLCA) patients suffer from malignant progression to life-threatening muscle-invasive bladder cancer (MIBC). Inflammation is a critical event in cancer development, but little is known about the role of inflammation in BLCA. In this study, the expression of the innate immune sensor AIM2 is much lower in high-grade BLCA and positively correlates with the survival rates of the BLCA patients. A novel AIM2 overexpressed BLCA model is proposed to investigate the impact of AIM2 on BLCA development. Mice inoculated with AIM2-overexpressed cells show tumor growth delay and prolonged survival compared to the control group. Meanwhile, CD11b+ cells significantly infiltrate AIM2-overexpressed tumors, and AIM2-overexpression in 5637 cells enhanced the inflammasome activation. In addition, oligodeoxynucleotide (ODN) TTAGGG (A151), an AIM2 inflammasome inhibitor, could abolish the elevation of AIM2-induced cleavage of inflammatory cytokines and pyroptosis. Orthotopic BLCA by AIM2-overexpressed cells exhibits a better response to Bacillus Calmette-Guérin (BCG) immunotherapy. Overall, AIM2 inflammasome activation can inhibit the BLCA tumorigenesis and enhance the therapeutic effect of BCG in BLCA. This study provides new insights into the anti-tumor effect of AIM2 inflammasome activation in BLCA and the immunotherapeutic strategy of BLCA development.

Histone H3K36me2 demethylase KDM2A promotes bladder cancer progression through epigenetically silencing RARRES3.

Epigenetic dysregulation contributes to bladder cancer tumorigenesis. H3K36me2 demethylase KDM2A functions as an important epigenetic regulator of cell fate in many types of tumors. However, its role in bladder cancer remains unknown. Here, we revealed a positive correlation between KDM2A gene copy number gain and upregulation of KDM2A mRNA expression in bladder cancer. Moreover, a super-enhancer (SE) driving KDM2A transcription was found in high-grade bladder cancer, resulting in a significantly higher expression of KDM2A mRNA compared to that in low-grade bladder tumors. KDM2A knockdown (KD) decreased the proliferation, invasion, and spheroid formation of high-grade bladder cancer cells and inhibited tumor growth in mouse xenograft models. Furthermore, we identified RARRES3 as a key KDM2A target gene. KDM2A suppresses RARRES3 expression via demethylation of H3K36me2 in the RARRES3 promoter. Intriguingly, RARRES3 KD attenuated the inhibitory effects of KDM2A depletion on the malignant phenotypes of high-grade bladder cancer cells. The combination of the KDM2A inhibitor IOX1 and the RARRES3 agonist all-trans retinoic acid (ATRA) synergistically inhibited the proliferation of high-grade bladder cancer cells, suggesting that the KDM2A/RARRES3 axis may be a promising therapeutic target for the treatment of high-grade bladder cancer.

The Efficacy of Combined Cisplatin and Nanoparticle Albumin-Bound Paclitaxel in a Stage IV Pancreatic Squamous Cell Carcinoma Patient With a Somatic BRCA2 Mutation: A Case Report.

Pancreatic squamous cell carcinoma (SCC) is a rare primary pancreatic malignancy with a poor prognosis. The median overall survival (OS) for metastatic setting is only 4 months and the optimal management remains poorly defined. In the present study, we report a 52-year-old female patient with stage IV primary SCC of the pancreas harboring a deleteous BRCA2 somatic mutation. After 10 cycles of chemotherapy of cisplatin combined with nanoparticle albumin-bound paclitaxel, metastatic lesions in the liver and lymph nodes achieved radiographic complete responses and pancreatic lesion shrank from 5.7 to 1.5 cm in diameter. The patient subsequently underwent a posterior radical antegrade modular pancreatosplenectomy with R0 resection and residual liver lesions were also resected. After 3 months, a tumor relapsed in the liver. She was then treated with olaparib combined with pembrolizumab and achieved stable disease on the liver lesion. The patient eventually died from cerebral hemorrhage with a long OS of 21 months. Our case demonstrated a favorable clinical activity and survival advantage of the combined cisplatin and nanoparticle albumin-bound paclitaxel, which might serve as a therapeutic option for the patient with BRCA-mutant pancreatic SCC.