Akihide Yoshimi

Translation initiation defines the protein output of the transcriptome, but its physiological dynamics in cells and tissues remain incompletely characterized. Here we develop TISCA2, an inhibitor-free method that captures eIF5B-associated initiating ribosomes to map translation initiation sites genome-wide. By directly identifying start sites rather than inferring translation from elongating-ribosome periodicity, TISCA2 improves the detection of short and overlapping translons. Productive translation of a subset of TISCA2-identified translons is supported by N-terminal proteomics and HLA-I immunopeptidomics. The arsenite-induced integrated stress response broadly redistributes start-site usage, reducing upstream initiation in favor of downstream initiation. TISCA2 further reveals tissue-specific initiation landscapes in vivo, with increased detection of downstream candidate start sites in the testis. Finally, we develop IncepTIS, a sequence-based deep learning model that predicts position-resolved and condition-dependent initiation landscapes. IncepTIS-guided optimization generates de novo mRNA 5′ leaders with stress-responsive initiation patterns. Together, TISCA2 and IncepTIS provide an experimental and computational framework for mapping, predicting and engineering translation initiation in mammalian cells and tissues.

Purpose: Spatially organized tertiary lymphoid structure (TLS)–like niches may provide prognostic and therapeutic information beyond individual immune mediators such as CXCL13 in bladder cancer. We evaluated the clinical and translational significance of multiplex immunofluorescence (mIF)–defined TLS-like structures (mTLS) in bladder cancer. Experimental Design: We analyzed 469 clinically annotated bladder cancer tumors using 41-plex mIF with the PhenoCycler platform. Bulk RNA sequencing, CIBERSORTx deconvolution, Cox modeling with CXCL13 as a continuous variable, and spatial neighborhood analysis were used to define mTLS-associated immune programs. Orthotopic bladder cancer mouse models treated with cisplatin and/or recombinant Cxcl13 followed by anti–PD-L1 blockade were analyzed by flow cytometry. Results: mTLS were identified in 22.2% of tumors and were not apparent on routine histopathology. mTLS positivity was associated with immune activation, improved survival, and favorable response patterns in a small PD-L1 blockade–treated subset. In Cox models, mTLS remained independently associated with outcomes, whereas continuous CXCL13 expression was not significant. Spatial profiling showed CXCL13/CXCR5-enriched B- and T-cell neighborhoods, including CXCL13-expressing and CXCR5-positive CD8+ T-cell subsets. In mouse models, cisplatin plus exogenous Cxcl13 was associated with lymphocyte infiltration, enrichment of CD8+ PD-1+ and CD8+ Cxcr5+ Cxcl13+ T-cell states, and sustained tumor control after PD-L1 blockade. Conclusions: mIF-defined mTLS identify a clinically relevant spatial immune architecture linked to favorable outcomes and therapeutic responsiveness in bladder cancer. These findings support prospective evaluation of mTLS as a spatial biomarker for risk stratification and treatment sequencing, with CXCL13/CXCR5 features interpreted as contextual companion markers rather than standalone determinants.

SUMMARY: NECTIN4 is the target of enfortumab vedotin (EV), but its biological role in bladder cancer (BC) and its relationship to EV plus PD-1 blockade remain unclear. Integrating clinical multi-omics, spatial and single-cell immune profiling, and experimental models, we identify NECTIN4 as a subtype-dependent determinant of tumor state and antitumor immunity. In basal BC cells, NECTIN4 suppressed inflammatory and mesenchymal programs. Consistently, NECTIN4-high basal/squamous muscle-invasive BCs showed favorable survival, higher checkpoint inhibitor response rates, and a coordinated CD8+ T cell-inflamed microenvironment with no comparable associations in luminal papillary tumors. In orthotopic models, murine Nectin4 promoted CD8+ T cell-dependent tumor control, whereas human NECTIN4 conferred EV sensitivity. EV also attenuated NECTIN4-dependent TIGIT binding. Conversely, NECTIN4-low models were preferentially sensitive to HDAC inhibition. NECTIN4 therefore couples antibody-drug conjugate targetability to a subtype-specific tumor–immune state permissive for checkpoint blockade, whereas the NECTIN4-low state defines a distinct inflammatory-mesenchymal state with an alternative therapeutic vulnerability. eTOC blurb: Nishimura et al. show that NECTIN4, the target of enfortumab vedotin, defines subtype-dependent tumor–immune states in bladder cancer. In basal/squamous tumors, NECTIN4 restrains inflammatory-mesenchymal programs and marks a CD8-rich microenvironment linked to checkpoint inhibitor response, whereas NECTIN4-low models show preferential sensitivity to HDAC inhibition. HIGHLIGHTS: NECTIN4 is a subtype-dependent regulator of tumor state, not just an ADC target NECTIN4-high Ba/Sq tumors exhibit a coordinated CD8-inflamed microenvironment Nectin4 drives CD8 T cell-dependent tumor control in immunocompetent mice NECTIN4 couples EV targetability to a checkpoint-responsive immune context

Human epidermal growth factor receptor 2 (HER2) has emerged as a potential therapeutic target in bladder cancer (BC), particularly with the advent of next‐generation antibody–drug conjugates (ADCs) like trastuzumab deruxtecan (T‐DXd). However, the complex relationship between HER2 protein expression, genetic status, and clinical outcomes remains incompletely understood. We conducted an integrated multi‐omics analysis of 445 BC cases (306 muscle‐invasive [MIBC] and 139 non‐muscle‐invasive [NMIBC]) from the OMPU‐NCC cohort, a uniform Japanese clinical database. The study utilized whole‐exome sequencing, RNA sequencing, and refined HER2 immunohistochemistry (IHC) scoring. HER2 IHC positivity (1+/2+/3+) was identified in 30.1% of the cohort. An additional 8.5% exhibited faint membrane staining despite being IHC 0, indicating that detectable HER2 membrane staining was present in approximately 38.5% of cases. HER2 IHC scores showed no prognostic value, whereas ERBB2 amplification, detected in 7.8% of cases, was associated with poorer survival and remained an independent adverse prognostic factor in multivariable analysis (HR, 1.76; 95% CI: 1.08–2.87). ERBB2 amplification was accompanied by frequent co‐amplification of genes within the 17q12 amplicon, including GRB7 , MIEN1 , and PGAP3 . Molecular subtyping revealed that ERBB2 amplification was enriched in the Luminal Unstable (LumU) subtype. In contrast, detectable HER2 membrane staining significantly correlated with high NECTIN4 expression, indicating an overlap between tumors expressing these two ADC targets. HER2 protein expression and ERBB2 amplification represent distinct features of BC. Their integrated assessment may improve molecular stratification and guide future biomarker‐driven studies.

Aberrant pre-mRNA splicing is a pervasive feature of cancer and an emerging therapeutic vulnerability. Recurrent mutations in core spliceosomal components, including SF3B1, SRSF2, U2AF1, and ZRSR2, are common in myeloid malignancies, while dysregulated splicing regulators and cis-acting splice-site alterations shape cancer-relevant isoform programs across solid tumors. Together with high transcriptional output, rapid proliferation, and oncogene-driven RNA-processing demand, these alterations can reduce the capacity of cancer cells to tolerate additional splicing perturbation, creating a therapeutic window for pharmacological splicing modulation. Multiple strategies are under investigation, including SF3B complex modulators, splicing kinase inhibitors, RBM39-directed molecular glues, PRMT/arginine-methylation-directed approaches, and selected splice-switching strategies. Early clinical experience indicates that pharmacodynamic modulation of splicing is achievable in patients, yet objective clinical benefit has been inconsistent. This reflects narrow therapeutic windows, incomplete concordance between peripheral-blood pharmacodynamic markers and tumor-tissue splicing perturbation, and the limited predictive value of mutation status alone. Rational combinations with apoptosis-targeted agents, oncogene-directed therapies, DNA-damaging agents, PARP inhibitors, and immunotherapies may offer a more effective route to clinical translation than maximal single-agent splicing inhibition. Continued progress will require more selective splicing-directed modalities, pharmacodynamic biomarkers that measure splicing perturbation in the relevant tumor or blood compartment, longitudinal mapping of genetic and tumor cell-state plasticity-driven resistance, and biomarker-defined combination trials to support expansion from hematologic malignancies into solid tumors.

Intraocular pressure (IOP), a major risk factor for glaucoma, follows a circadian rhythm with nocturnal increases driven by norepinephrine (NE) from the superior cervical ganglion. This rhythm depends on aqueous humor (AH) dynamics, particularly the outflow through the trabecular meshwork (TM). Herein, we investigated its underlying regulatory mechanisms in the TM because disruption of IOP rhythm increases the risk of glaucoma. Comprehensive gene expression analysis of human TM cells and mouse eyes uncovered NE-upregulated 18 genes, including the small GTPase RHOB. Promoter assays revealed that RHOB is regulated via a cAMP-responsive element. RHOB deficiency enhanced TM phagocytosis and reversed NE-induced suppression of permeability, whereas RHOB overexpression had the opposite effect. In mice, RHO and ROCK inhibitors reduced both nocturnal and NE-induced IOP elevations. These findings suggest that NE elevates IOP by inhibiting TM phagocytosis via RHOB, identifying RHOB as a key regulator of IOP rhythm and a potential target for glaucoma treatment.

Bladder-preserving trimodality therapy (TMT) incorporating concurrent chemoradiotherapy (CRT) provides a curative-intent alternative to radical cystectomy for muscle-invasive bladder cancer (MIBC), yet its efficacy is frequently limited by intrinsic treatment resistance, the molecular basis of which remains poorly defined. To address this, we performed bulk transcriptomic profiling of pretreatment tumors from 179 patients uniformly treated with bladder-preserving CRT and systematically integrated gene expression data with tumor immune features and clinical outcomes. We identified a ferroptosis-suppressive transcriptional signature (FSS) associated with a distinct resistance-associated tumor state that independently stratified radiographic progression-free survival and overall survival following CRT. FSS-high tumors were characterized by inferior outcomes, enrichment of basal/squamous and immune-excluded phenotypes, and reduced intratumoral immune infiltration, whereas FSS-low tumors preferentially exhibited luminal unstable and immune-inflamed features. Consistent with clinical observations, a ferroptosis-suppressive transcriptional program was recapitulated in CRT-resistant bladder cancer cell line models. Genome-wide CRISPR/Cas9 loss-of-function screening further identified core ferroptosis suppressors as functionally relevant dependencies specifically under irradiation stress, and pharmacologic induction of ferroptosis effectively restored radiosensitivity in otherwise resistant cells. Together, these findings support ferroptosis suppression as a biologically relevant resistance-associated state that mechanistically links tumor-intrinsic transcriptional programs to immune contexture and therapeutic vulnerability and provide a translational framework for improved risk stratification and future treatment refinement in bladder-preserving therapy for MIBC.

Alternative pre-mRNA splicing generates extensive transcript diversity, yet the regulatory code that determines how splicing decisions are encoded across the transcriptome remains poorly defined. Splicing outcomes are controlled by combinatorial RNA-binding protein (RBP) interactions and positional context, but how these features are integrated at the transcriptome scale remains unclear. CLIP-based approaches have mapped RBP binding, but directly comparable endogenous maps across multiple RBPs are lacking, limiting inference of global regulatory principles. Here we introduce SCALE-CLIP, an endogenous CLIP framework that integrates CRISPR-Cas9-mediated epitope tagging with long-read-guided read attribution to generate directly comparable RBP binding maps across splicing-regulatory factors. Applied to 23 RBPs, SCALE-CLIP expanded endogenous RBP coverage and, across benchmarked shared factors, increased peak recovery by a median of 12.2-fold relative to ENCODE eCLIP while preserving specificity and reproducibility. We define a transcriptome-wide positional and combinatorial code for alternative splicing, in which binding position is a primary determinant of regulatory outcome: SRSF binding within alternative exons promotes inclusion, whereas binding on flanking exons drives exon skipping. Higher-order SRSF occupancy further tunes this code, buffering exon inclusion when centered on alternative exons but reinforcing repression when distributed across flanking exons. We also show that m ⁶ A provides an epitranscriptomic layer that locally enhances SRSF binding and is associated with increased exon inclusion. Together, these results establish a multi-layered RNA-binding logic in which binding position, combinatorial RBP architecture and RNA modification jointly shape splicing outcomes, providing a framework for rational interpretation and modulation of alternative splicing.