Aberrant alternative splicing (AS) events have been implicated in cancer progression; however, their role in metastatic renal cell carcinoma (mRCC) remains underexplored. This study aims to identify AS events associated with clinical benefits from immune checkpoint inhibitors and targeted therapies in mRCC. We conducted a retrospective analysis on 101 patients with mRCC who received systemic therapy and underwent RNA sequencing. Patients were divided into subgroups based on ICIs (alone or in combination) and targeted therapies. Responders and non-responders were classified according to Response Evaluation Criteria in Solid Tumors V.1.1 criteria. Differential gene expression and splicing analyses were performed between responders and non-responders in each cohort. Novel AS events were analyzed for their potential to generate peptide neoantigens through major histocompatibility complex (MHC) class I binding predictions. Outlier splicing analysis identified 10 aberrant splice events specific to mRCC. AS analysis revealed 461 differentially spliced events between responders and non-responders in the ICI cohort and 253 in the targeted therapy cohort, with intron retention as the predominant motif. Thirteen unique AS events were enriched in responders, including PTPN6 and ACTN1. Predictive neoantigen analysis identified high MHC class I binding potential in peptides from AS events in IFFO1 and ZNF692. High splice burden was linked to an immunogenic tumor microenvironment, characterized by enriched antigen processing and adaptive immune responses. This study provides a comprehensive analysis of AS events in mRCC, highlighting intron retention as potential biomarkers for treatment response. Identified AS-derived neoantigens may serve as potential targets for adoptive cell therapy strategies.

Nivolumab plus ipilimumab (COMBO) is the standard treatment for asymptomatic melanoma brain metastases (MBM), but current guidelines do not provide specific recommendations for treatment discontinuation in responding patients. This study aimed to evaluate outcomes after COMBO discontinuation within 24 months and the role of continuing treatment beyond 24 months. Patients with MBM treated with COMBO who discontinued treatment within 24 months for reasons other than disease progression or continued beyond this time point were retrieved. Overall survival (OS), objective response, progression-free survival (PFS) and toxicities were analyzed. 465 patients were included: 392 discontinued COMBO within 24 months, while 73 continued beyond 24 months. Treatment was discontinued due to complete response (CR, n=47), partial response (PR, n=45), stable disease (SD, n=12), toxicity after SD (n=59), toxicity after CR (n=99), or toxicity after PR (n=130). At multivariable analysis, the line of treatment (>first vs first: HR 2.65 (1.62-4.32)), the immune-related adverse events (irrespective of anti-tumor necrosis factor-alpha) (HR 0.18 (0.07-0.42)); COMBO discontinuation after CR (HR 0.15 (0.05-0.40)), or PR (HR 0.08 (0.03-0.26)), as well as stopping due to toxicity after CR (HR 0.14 (0.07-0.27)) or PR (HR 0.51 (0.32-0.82)), were associated with OS. Notably, at a median follow-up of 51 months (IQR 31-70), patients with CR/PR who discontinued COMBO within 24 months had PFS and OS comparable to those who continued treatment beyond this time point. 4-year OS exceeded 83% in patients discontinuing COMBO after CR, PR, or toxicity following CR, compared with 66.4% in those discontinuing due to toxicity after PR; median PFS was not reached in the former groups but was 18.6 months in the toxicity after PR group. Discontinuation of COMBO within 24 months appears safe in patients with CR and in selected cases of PR, with no survival disadvantage versus prolonged therapy.

Immune checkpoint blockade (ICB) unleashes antitumor immunity but frequently provokes enduring endocrine toxicities. We hypothesize that ICB accelerates adrenal aging by establishing chronic low-level inflammation within the adrenal cortex, with targeting vulnerability of the zona reticularis. Integrating a recently published human multiorgan aging proteome atlas and primate adrenal aging study with survivorship data after ICB therapy, we propose a testable signaling cascade: ICB-amplified interferon gamma (IFNγ)/ tumor necrosis factor (TNF)/ interleukin-1 signaling activates nuclear factor kappa B (NF-κB)/signal transducer and activator of transcription 1 (STAT1), suppressing sterol regulatory element-binding protein 2 (SREBP2)-low-density lipoprotein receptor (LDLR)-mediated cholesterol uptake; concurrent mitochondrial/endoplasmic reticulum stress drives proteome-transcriptome decoupling, loss of cytochrome b5 type A (CYB5A), and impaired cytochrome P450 family 17 subfamily A member 1 (CYP17A1) 17,20-lyase activity; inflammatory transcriptional repression of sulfotransferase family 2A member 1 (SULT2A1) with proteostasis decay reduces dehydroepiandrosterone (DHEA) sulfation. The net result is a persistent fall in DHEA/DHEA sulfate (DHEAS) with comparatively preserved cortisol-mirroring natural adrenal aging. We advocate prospective measurement of DHEAS, DHEA, adrenocorticotropic hormone (ACTH), and cortisol at baseline, during therapy, end of therapy, and 6-24 months post-therapy; if early DHEAS decline is confirmed, targeted interventions including DHEA replacement or glucocorticoid receptor antagonism warrant evaluation. This framework reframes certain endocrine immune-related adverse events as "accelerated organ aging," with implications for risk stratification, toxicity prevention, and survivorship care.

Breast cancer brain metastasis (BCBrM) remains one of the most lethal manifestations of breast cancer. Its response to immunotherapy is severely limited by the blood-brain barrier, which restricts immune cell infiltration and antigen presentation, thereby creating an immunosuppressive microenvironment. To overcome these barriers, recent studies have focused on novel immune checkpoints, including the Lymphocyte-Activated Gene 3–Galectin 3 (LAG3–LGALS3) and T-Cell Immunoreceptor with Ig and ITIM Domains–Nectin Cell Adhesion Molecule 2 (TIGIT–NECTIN2) axes, as well as on the reprogrammed metastatic ecosystem driven by immunosuppressive cells such as Forkhead Box P3-positive (FOXP3⁺) Regulatory T (Treg) cells, Lysosomal-Associated Membrane Protein 3-positive (LAMP3⁺) tolerogenic dendritic cells (DCs), C-C Motif Chemokine Ligand 18-positive (CCL18⁺) M2-like macrophages, Regulator of G-Protein Signaling 5-positive (RGS5⁺) cancer-associated fibroblasts (CAFs), Galectin 1-positive (LGALS1⁺) and TANK-Binding Kinase 1-positive (TBK1⁺) microglia, and phosphorylated Signal Transducer and Activator of Transcription 3-positive (pSTAT3⁺) reactive astrocytes. In addition, targeted inhibition of tumor-derived N-acetyltransferase 8-like (NAT8L) and metabolites N-Acetylaspartate (NAA), suppression of the N-Methyl-D-Aspartate Receptor (NMDAR) signaling pathway in tumor cells, and interventions against γ-Aminobutyric Acid (GABA)ergic reprogramming in BCBrM cells. Moreover, targeted interventions against distinct immune escape pathways—such as the Ubiquitin-Conjugating Enzyme E2T (UBE2T)/Cell Division Cycle 42 (CDC42)/Cluster of Differentiation 276 (CD276) and C-C Motif Chemokine Ligand 2–C-C Motif Chemokine Receptor 2/C-C Motif Chemokine Receptor 4 (CCL2–CCR2/CCR4) axes—have shown promise in reshaping the immune microenvironment and enhancing the efficacy of conventional immunotherapy. Collectively, this perspective outlines evolving strategies in immune checkpoint modulation, cellular ecosystem reprogramming, and neuroimmune intervention, providing a forward-looking framework to enhance the efficacy of immunotherapy in BCBrM.

BackgroundInsufficient T-cell infiltration limits the effectiveness of immunotherapy in sarcoma, yet the tumor-intrinsic mechanisms that govern immune exclusion remain poorly defined.MethodsBy integrating patient-derived ex vivo sarcoma spheroids with autologous expanded tumor-infiltrating lymphocytes and an in vivo metastatic osteosarcoma model, antitumor immune regulation by histone modifications was examined.ResultsHistone H3 lysine 27 acetylation (H3K27ac) was identified as a key regulator of CD8+ T-cell infiltration in osteosarcoma and other bone and soft-tissue sarcomas. Pharmacological elevation of H3K27ac by the histone deacetylase 1/3 inhibitor entinostat promotes CD8+ T-cell activation, cytotoxicity, and the recruitment of CD8+CD103+ tissue-resident memory T cells. Mechanistically, these immune-boosting effects are triggered by a Hippo pathway switch, in which yes-associated protein 1 (YAP1) is suppressed, and vestigial-like family member 3 (VGLL3) is induced, thereby modulating transcription towards an immune-responsive state. Furthermore, we identified that VGLL3/CD103 signatures predict a response to anti-programmed cell death protein-1 (PD-1) treatment in patients with sarcoma, and that combining H3K27ac induction with anti-PD-1 further augments T cell-mediated killing in ex vivo autologous patient-derived spheroid models.ConclusionsOur findings reveal an epigenetic-Hippo-immunomodulatory axis in osteosarcoma that also extends to other sarcomas, providing a rationale for incorporating epigenetic preconditioning with immunotherapy to improve patient outcomes and pointing towards novel biomarkers for treatment guidance.

Antigen-specific CD8+ T cells can be in a stem-like programmed cell death protein-1 (PD-1)+TCF-1+ differentiation state that progresses into terminal exhaustion in cancer and chronic infection. These stem-like cells are important, since they are the responders to PD-1 targeted immunotherapy and a potential resource for antitumor immunity. We use a mouse vaccination model to delineate by spectral flow cytometry and single-cell RNA sequencing the effects of CD4+ T-cell help during priming on the differentiation fate of stem-like CD8+ T cells. We use bioinformatic analysis to extrapolate our data to mouse models of cancer and chronic infection. We next explore CD8+ T-cell differentiation states and delivery of CD4+ T-cell help in the immunogenic MC38 tumor model. Upon vaccination in the absence of help signals, stem-like CD8+ T cells do not further differentiate and accumulate in the draining lymph node. When help signals are delivered, stem-like CD8+ T cells proliferate and differentiate into circulating cytotoxic effector cells. Stem-like CD8+ T cells raised by vaccination in presence or absence of CD4+ T-cell help have an identical transcriptome, which they share with stem-like CD8+ T cells defined in mouse models of cancer and chronic infection. The immunogenic MC38 tumor harbors endogenous helper epitopes, but primes stem-like CD8+ T cells rather than helped cytotoxic effectors. Therapeutic vaccination with endogenous helper epitopes does not improve MC38 tumor control. Intratumoral expression of strong, exogenous helper epitopes as present in our vaccine improves tumor control, but does not efficiently convert stem-like tumor-specific CD8+ T cells into helped cytotoxic effectors. Our data argue that stem-like CD8+ T cells are helpless cells that lie at the bifurcation point of CD8+ T-cell effector and exhaustion trajectories. Even though the immunogenic MC38 tumor expresses helper epitopes, it primarily raises stem-like CD8+ T cells, indicating that help delivery is impaired in this tumor context. Promoting the efficient delivery of help signals to stem-like tumor-specific CD8+ T cells to drive their expansion and differentiation into cytotoxic effectors is therefore an important therapeutic challenge in cancer and other conditions that lead to T-cell exhaustion.

Recent studies have indicated that Janus kinase inhibitors (JAKi) exert context-dependent immunomodulatory effects in cancer immunotherapy. In this rapid response, we comment on the prospective cohort study by Habib et al, which evaluates JAKi for steroid-refractory or life-threatening immune-related adverse events (irAEs) associated with immune checkpoint inhibitors (ICIs). The study demonstrated a clinical remission rate of 70.8%, with notable activity in myocarditis and inflammatory arthritis. Although JAK inhibition has historically raised concern due to the central role of JAK-signal transducer and activator of transcription (STAT) signaling in interferon-mediated antitumor immunity, emerging clinical data indicate more nuanced effects influenced by timing, dosing, and immune context. This framework may help explain the observed efficacy of JAKi in controlling severe irAEs without an apparent excess of opportunistic infections. We also emphasize unresolved safety considerations, including thromboembolic risk and disease progression during follow-up. Given the clinical heterogeneity of the cohort and the limited oncologic detail, further prospective studies with stratified and time-to-event analyses are needed to better define the role of JAK inhibition in balancing irAE control and antitumor immunity.

Although immune checkpoint inhibitors (ICIs) have long half-lives, preclinical and retrospective clinical studies across multiple tumor types suggest that the time-of-day of ICI infusion may influence therapeutic efficacy by aligning initial drug exposure with circadian peaks in T-cell responsiveness. The immunological basis of this phenomenon and its clinical relevance in hepatocellular carcinoma (HCC) remains unknown. We followed patients with advanced HCC receiving ICI therapy at Johns Hopkins from 2021 to 2025, classifying them into a morning (first treatment before 12:00 hours) or afternoon (first treatment after 12:00 hours) group. We assessed clinical outcomes and compared immunological responses from baseline to early-on-treatment by profiling peripheral blood mononuclear cells using cytometry by time-of-flight and plasma cytokines using a 39-plex Luminex assay. Our cohort included 84 patients, 39 of whom received their first infusion in the morning. There were no statistically significant differences in baseline demographic or clinical characteristics between patients initiating therapy in the morning versus afternoon. The morning group had superior progression-free survival (multivariable HR 0.50, 95% CI 0.30 to 0.84, p<0.01) and higher odds of treatment response (multivariable OR 3.26, 95% CI 1.08 to 10.90, p<0.05), with no significant increase in immune-related adverse events. The timing of subsequent infusions after the first dose had no impact on outcomes. Immunological responses diverged after the initial dose, with morning-treated patients showing reduced interleukin (IL)-6 levels (p<0.01) and greater expansion of cytotoxic central memory CD8+ T cells (p=0.01) as well as cytotoxic effector and effector memory CD8+ T cells (p=0.06). Morning first-dose infusion of ICIs in HCC was associated with improved clinical outcomes and distinct immune responses, including reduced IL-6 signaling and expansion of cytotoxic central memory CD8+ T cells. These findings suggest that the timing of the initial infusion can imprint an immunological program that shapes subsequent antitumor immunity, providing a mechanistic rationale for strategically scheduling ICI administration.

BackgroundOncolytic virotherapy represents a promising anticancer strategy by combining direct tumor lysis with in situ immune activation. However, its efficacy remains limited in immune-cold tumors, which are characterized by poor T-cell infiltration and an immunosuppressive microenvironment. Although engineering oncolytic viruses to deliver chemokines has been explored to modulate immune recruitment, most existing approaches activate only a single immune axis. Senecavirus A (SVA), a tumor-selective RNA virus with high genetic stability and flexible transgene capacity, offers an attractive platform for coordinated intratumoral delivery of immune payloads.MethodsWe engineered recombinant SVA vectors to achieve co-expression of CXCL11 and vXCL1 within tumor, aiming to synergistically recruit both T cells and cross-presenting dendritic cells and remodel the tumor immune landscape. In vitro and in vivo assays were conducted to assess viral properties (stability, replication kinetics, gene expression) and cytotoxicity against B16-F10 melanoma cells in different stages, with tumor burden, immune cell infiltration, and mouse survival in C57BL/6 mice analyzed to evaluate the overall therapeutic efficacy.ResultsSignal-peptide deletion significantly improved the genetic stability of transgenes and enhanced intratumoral payload retention, without compromising viral fitness. In murine models, CXCL11-expressing virus monotherapy achieved enhanced tumor control, prolonged survival, and provided preliminary evidence suggestive of protection on tumor rechallenge. Furthermore, the combination of CXCL11 and vXCL1 further augmented dendritic cell activation, promoted CD8+ T cell infiltration, and shifted macrophages toward an M1-polarized phenotype, while sustaining higher intratumoral viral loads. These coordinated immune changes led to deeper and more durable tumor regression.ConclusionsOur findings validate the combination of CXCL11- and vXCL1-armed SVA as a potent immunovirotherapy strategy and propose a design principle for multistage, multigene intervention: concurrently targeting leukocyte recruitment, antigen presentation, and effector activation provides complementary mechanisms that synergistically amplify therapeutic efficacy.