Intratumoral Tregs are key mediators of cancer immunotherapy resistance, including anti–programmed cell death (ligand) 1 [anti–PD-(L)1] immune checkpoint blockade (ICB). The mechanisms driving Treg infiltration into the tumor microenvironment (TME) and the consequence on CD8 ⁺ T cell exhaustion remain elusive. Here, we report that heat shock protein gp96 (also known as GRP94) was indispensable for Treg tumor infiltration, primarily through the roles of gp96 in chaperoning integrins. Among various gp96-dependent integrins, we found that only LFA-1 (αL integrin), and not αV, CD103 (αE), or β7 integrin, was required for Treg tumor homing. Loss of Treg infiltration into the TME by genetic deletion of gp96/LFA-1 potently induced rejection of tumors in multiple ICB-resistant murine cancer models in a CD8 ⁺ T cell–dependent manner, without loss of self-tolerance. Moreover, gp96 deletion impeded Treg activation primarily by suppressing IL-2/STAT5 signaling, which also contributed to tumor regression. By competing for intratumoral IL-2, Tregs prevented the activation of CD8 ⁺ tumor-infiltrating lymphocytes, drove thymocyte selection-associated high mobility group box protein (TOX) induction, and induced bona fide CD8 ⁺ T cell exhaustion. By contrast, Treg ablation led to striking CD8 ⁺ T cell activation without TOX induction, demonstrating clear uncoupling of the 2 processes. Our study reveals that the gp96/LFA-1 axis plays a fundamental role in Treg biology and suggests that Treg-specific gp96/LFA-1 targeting represents a valuable strategy for cancer immunotherapy without inflicting autoinflammatory conditions.

Tumor-associated macrophages (TAMs) play a crucial role in the tumor microenvironment, yet the roles and mechanisms of TAMs in inflammation-associated oncogenesis remain enigmatic. We report that protein canopy homolog 2 (CNPY2) functions as a novel TLR4 regulator, promoting cytokine production in macrophages. CNPY2 binds directly to TLR4. Cnpy2 deficiency reduces cell surface expression of TLR4, nuclear translocation of NFκB and cytokine production in macrophages. Macrophage-specific CNPY2 deficiency significantly decreases cytokine production in macrophages and reduces hepatocarcinogenesis in a diethylnitrosamine (DEN)-induced liver cancer model. RNA-sequencing analysis revealed Cnpy2 knockout decreased the mRNA level and cell surface expression of two VEGF receptors, Flt1 and Kdr, compared to those in WT counterparts, resulting in inhibition of macrophage tumor infiltration. Cnpy2 knockout inhibits NFκB2/p52-mediated transcription of Flt1 and Kdr in macrophages. These findings demonstrate that CNPY2 regulates macrophages in both inflammation and hepatocarcinogenesis and may serve as a therapeutic target for cancer.

Canopy FGF signaling regulator 2 (CNPY2) has emerged as a crucial player in cancer development by promoting cell proliferation, tissue repair, and angiogenesis. This review synthesizes the current understanding of CNPY2’s role in solid tumors, particularly renal cell carcinoma, prostate cancer, hepatocellular carcinoma, and non-small-cell lung cancer. CNPY2 modulates key pathways such as p53, MYLIP, NF-κB, and AKT/GSK3β, thereby driving tumor growth and progression. In renal cell carcinoma, CNPY2 paradoxically promotes tumor growth through p53 upregulation, while in hepatocellular carcinoma, CNPY2 drives cell cycle progression via p53 destabilization. In prostate cancer, it enhances tumor progression by stabilizing androgen receptors through MYLIP interaction, and in non-small-cell lung cancer, it contributes to chemoresistance and metastasis through NF-κB and AKT/GSK3β signaling. Additionally, CNPY2 influences the tumor microenvironment, impacting immune function and metastatic potential. As a potential biomarker, CNPY2 shows promise for cancer detection and prognosis, particularly when used in combination with other markers. Early therapeutic strategies, including siRNA and miRNA approaches, are under exploration, though challenges remain due to CNPY2’s expression in normal tissues and potential off-target effects. This review underscores the need for further research to fully elucidate CNPY2’s oncogenic mechanisms and develop targeted therapies. Improved understanding of CNPY2’s diverse roles may lead to novel diagnostic and therapeutic approaches in solid tumors.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal tract, driven by genetic, environmental, and immune system factors. However, its exact mechanisms remain unclear. Here, we demonstrate that CNPY2 plays a critical role in colitis by modulating macrophage activity. Mice with whole-body Cnpy2 knockout (KO) exhibited significantly reduced dextran sodium sulfate (DSS)-induced colitis compared to wild-type controls. Cnpy2 KO mice showed less mucosal barrier disruption and fewer lamina propria macrophages (LPMφs) following DSS treatment. Proinflammatory cytokine production was also diminished in the colons of Cnpy2 KO mice. Furthermore, Cnpy2 KO macrophages generated markedly lower levels of reactive oxygen species (ROS), partly through CHOP regulation. Notably, treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) completely abolished DSS-induced colitis in Cnpy2 KO mice. Thus, CNPY2 exacerbates DSS-induced colitis primarily through macrophage-specific effects, with ROS upregulation being central to its pathogenic role.

Background/Objectives: Enfortumab vedotin plus pembrolizumab (EV+pembro) is the standard first-line treatment for metastatic urothelial carcinoma (mUC), yet no validated on-treatment biomarker identifies patients deriving a durable benefit. We evaluated whether circulating tumor DNA (ctDNA) clearance is associated with response and survival with this regimen alone. Methods: This single-center retrospective study included 25 patients with mUC receiving first-line EV+pembro. A tumor-informed assay (Signatera) measured ctDNA longitudinally. Responses were assessed by RECIST 1.1. Progression-free survival (PFS) and overall survival (OS) hazard ratios (HRs) were estimated by Cox regression and, to address immortal time bias, with clearance as a time-dependent covariate. Results: Fifteen of 25 patients (60%) achieved ctDNA clearance (ever undetectable). The objective response rate was 76% overall and 100% (15/15) with clearance versus 40% (4/10) without (p = 0.0012). Clearance was associated with longer PFS (HR 0.16, 95% CI 0.05–0.54; p = 0.003) and OS (HR 0.08, 95% CI 0.01–0.68; p = 0.021), with similar Firth penalized HRs (0.17 and 0.11). In the time-dependent analysis, the associations were attenuated (PFS HR 0.35, 95% CI 0.09–1.15, p = 0.085; OS HR 0.14, 95% CI 0.01–0.84, p = 0.029; profile likelihood CIs). A post-nadir ctDNA rise preceded progression or death by a median of 95 days in four patients (death in two). Conclusions: In this first-line EV+pembro cohort, all patients who cleared ctDNA responded, and clearance was associated with longer survival, although this association was attenuated in the time-dependent analysis. These hypothesis-generating findings support the prospective evaluation of ctDNA-guided monitoring.