Abstract Introduction IL-2 is central to CD8 T cell responses and signals via JAK-STAT5, RAF-ERK-MAPK, and PI3K-AKT pathways, but how STAT5-dominant output is enforced from this shared receptor is unclear. We hypothesized that uncharacterized IL-2-induced, STAT5-regulated E3 ubiquitin ligases sculpt this signaling hierarchy to optimize antiviral CD8 T cell function. Methods RNA-seq of human CD8 T cells stimulated with IL-2 ± JAK inhibition plus STAT5 ChIP-seq identified IL-2/STAT5-regulated ubiquitin genes. Rnf144a-/- mice and mixed bone marrow chimeras were used to define CD8-intrinsic roles during influenza infection. RNF144A localization and substrates were mapped by biochemical and imaging assays, and whole-blood transcriptomes from patients with moderate or severe influenza were analyzed to relate RNF144A expression and gene signatures to clinical severity. Results IL-2 broadly remodeled the T cell ubiquitin program, with RNF144A emerging as the most strongly induced STAT5-bound E3 ligase. RNF144A localized to the plasma membrane, associated with IL-2Rβ and STAT5, and enhanced STAT5 recruitment and phosphorylation, sustaining STAT5-dependent transcription. In parallel, RNF144A acted as a bona fide E3 ligase that directly polyubiquitinated RAF1 for degradation, reducing ERK activation and thereby preserving JAK-STAT5 dominance over RAF-ERK-MAPK output. CD8 T cells from Rnf144a-/- mice showed impaired IL-2-induced effector gene expression, degranulation, and cytokine production, and CD8-intrinsic deficiency in mixed chimeras reduced antigen-specific responses and worsened weight loss and lung inflammation after influenza infection. In human influenza, RNF144A expression was reduced in severe disease, inversely correlated with an ERK-MAPK target gene signature, and discriminated severe from moderate cases with performance comparable to established severity markers. Conclusion RNF144A is an IL-2-STAT5-induced E3 ligase that enforces STAT5-dominant signaling and limits viral immunopathology. Funding Source N/A Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

The immunosuppressive transmembrane protein PD-L1 was shown to traffic via the multivesicular body (MVB) and to be released on exosomes. A high-content siRNA screen identified the endosomal sorting complexes required for transport (ESCRT)-associated protein ALIX as a regulator of both EGFR activity and PD-L1 surface presentation in basal-like breast cancer (BLBC) cells. ALIX depletion results in prolonged and enhanced stimulation-induced EGFR activity as well as defective PD-L1 trafficking through the MVB, reduced exosomal secretion, and its redistribution to the cell surface. Increased surface PD-L1 expression confers an EGFR-dependent immunosuppressive phenotype on ALIX-depleted cells. An inverse association between ALIX and PD-L1 expression was observed in human breast cancer tissues, while an immunocompetent mouse model of breast cancer revealed that ALIX-deficient tumors are larger and show an increased immunosuppressive environment. Our data suggest that ALIX modulates immunosuppression through regulation of PD-L1 and EGFR and may, therefore, present a diagnostic and therapeutic target for BLBC.

A, Modulation of expression of PSMA protein in CWR22 and 22RV1 cells by inhibitors with maximum upregulation induced by DS-7423 (3.4- and 5.6-fold, respectively). B, Detection of PSMA mRNA after treatment with DS-7423. Results are shown as mean with SE (n = 3; ***, P < 0.001).

INTRODUCTION:Toll-like receptor 9 (TLR9) is primarily expressed in human dendritic and B cells and recognizes double-stranded DNA motifs from pathogens to initiate an inflammatory response. Recent studies have revealed TLR9s' involvement beyond its conventional role in the immune response, notably during the tumorigenesis of various cancers such as head and neck, cervical, and ovarian cancers. METHODS:In this study patient biopsies of breast cancer tumors and normal breast epithelium were analyzed by immunohistochemistry to examine TLR9 expression. The study also investigated downregulation in transformed breast cancer cell lines compared to untransformed breast epithelial cells by analyzing gene or protein expression, including TLR9, IL-6, CCL2, CXCL1, and GM-CSF. MDA-MB-361 cells were engineered to express exogenous TLR9, and the effects on colony growth and senescence were assessed using colony formation assays, senescence staining, cytokine analysis, and flow cytometry. RESULTS:TLR9 levels in breast cancer tumors were significantly reduced compared to normal breast tissue epithelium. This downregulation was also observed in several transformed breast cancer cell lines compared to untransformed breast epithelial cell lines. Furthermore, MDA-MB-361 breast cancer cells expressing exogenous TLR9 exhibited reduced colony growth and an increase in the senescence marker IL-6, pro-inflammatory cytokine CCL2, CXCL1 chemokine; and growth factor GM-CSF. CONCLUSION:These findings support TLR9's regulatory role in mitigating breast cancer and highlight its critical connection between the innate immunity and tumor cell growth.

A, Representative images of clonogenic survival assays in CWR22 cells treated with CPCCOEt, VCC185369, and lapatinib alone or in combination with DS-7423 inhibitor. B, Quantification of clonogenic survival assays from A. Results are shown as mean with SE (n = 3; ***, P < 0.001). C, Tumor volume growth curves of CWR22 xenografts for the indicated treatments. Results are shown as mean with SE (n = 4–5; #, P = 0.06; **, P < 0.01). For those individuals that were culled before the end of the experiment due to ethical limits, a growth constant was calculated as (log fold change), and the tumor volume was estimated to obtain the average.

A, qRT-PCR analysis of GRM1 gene expression in CWR22 and 22RV1 cells. Results are shown as mean with SE (n = 3; *, P < 0.05; **, P < 0.01). B, Expression of HER2, HER3, and PSMA in control and GRM1-depleted CWR22 cells. C and D, Quantification of HER2 and PSMA expression in cells from B. Results are shown as mean with SE (n = 3; *, P < 0.05; **, P < 0.01). E, Expression of of HER2, HER3, and PSMA after treatment with inhibitor alone or in combination. F and G, Quantification of expression of HER2 and PSMA presented in E. Results are shown as mean with SE (n = 3).

Expression profile for indicated proteins in prostate cancer cells after treatment with PI3K, mTOR, or dual inhibitors for 48 hours. A, DS-7423 increased expression of HER2 (4.5-fold in CWR22, 7.3-fold in 22RV1, 1.8-fold in PC3, and 1.2-fold in Shmac5 cells), HER3 (5-fold in LNCap, 1.3-fold in CWR22, 18-fold in PC3, and 4.5-fold in Shmac5 cells), and AR (6.1-fold in LNCaP). B, Immunofluorescence images show the DS-7423–induced upregulation of HER2 (green) and HER3 (red) in CWR22 and 22RV1 cells. Nucleus stained with Hoechst33342 (blue); scale bar, 50 μm.

Model proposed describing the PSMA-mGluR1-HER2 mechanism of resistance to dual PI3K-mTOR inhibitor DS-7423 (created with BioRender.com). HER2/X represents the HER2-containing receptor protein complex (X = EGFR or HER3 in Supplementary Fig. S1E and S1F). A feedback mechanism that is activated in response to PI3K/mTOR inhibition involves upregulation of ErbB/HER protein (HER2 or HER3) according to the genetically diverse prostate cancer cell lines we have tested. Downstream effects include a PSMA increase upon DS-7423 treatment that is partially dependent on HER2 signaling. Targeting the PI3K/mTOR pathway has unraveled a complex relationship between PSMA increase and HER2 upregulation via mGluR1 activity (Fig. 4B).

Chemotherapy, the standard of care treatment for cancer patients with advanced disease, has been increasingly recognized to activate host immune responses to produce durable outcomes. Here, in colorectal adenocarcinoma (CRC) we identify oxaliplatin-induced Thioredoxin-Interacting Protein (TXNIP), a MondoA-dependent tumor suppressor gene, as a negative regulator of Growth/Differentiation Factor 15 (GDF15). GDF15 is a negative prognostic factor in CRC and promotes the differentiation of regulatory T cells (Tregs), which inhibit CD8 T-cell activation. Intriguingly, multiple models including patient-derived tumor organoids demonstrate that the loss of TXNIP and GDF15 responsiveness to oxaliplatin is associated with advanced disease or chemotherapeutic resistance, with transcriptomic or proteomic GDF15/TXNIP ratios showing potential as a prognostic biomarker. These findings illustrate a potentially common pathway where chemotherapy-induced epithelial oxidative stress drives local immune remodeling for patient benefit, with disruption of this pathway seen in refractory or advanced cases.

Supplementary video showing fluorescence-guided resection of a subcutaneous model of Neuroblastoma injected with anti-GD2 fluorescence probes

In vivo validation of tumor uptake of anti–GD2-IR800 and anti–GD2-IR12 using a commercially available clinical NIR-I imaging device. A, Comparison of fluorescent images of the unexposed tumors at different time points postinjection of either anti–GD2-IR800 or anti–GD2-IR12. The laser boost setting of the EleVision IR Platform was set to arbitrary units for 24, 48, 72, 96 hours and exposed anti–GD2-IR12 images, respectively. B, Bar chart showing MFI of the unexposed tumors post anti–GD2-IR800 and anti–GD2-IR12 injections. Quantification was performed using the EleVision software. C, Bar chart of the TBR. Error bars are calculated from the SD across 5 points manually selected in each ROI. Anti–GD2-IR800, n = 2 at all time points; anti–GD2-IR12, n = 2 at 24 hours, n = 1 at 48, 72, and 96 hours. D, Images showing stages of tumor excision after injection of anti–GD2-IR800 under white light observation and NIR-I fluorescence. A residual tumor (3 × 5 mm) was identified on fluorescence imaging and subsequently excised. E, Histopathologic evaluation of both the main tumor and residual tissue, confirming the presence of viable neuroblastoma.

Biological evaluation of anti–GD2-IR800 and anti–GD2-IR12 using a preclinical NIR-I imaging system in vivo. A, IVIS Spectrum images of the exposed tumors at 24, 48, 72, and 96 hours postinjection of anti–GD2-IR800 (n = 3) and anti–GD2-IR12 (n = 3) in mice bearing subcutaneous LAN-1 neuroblastoma tumors. White dotted lines show the ROIs drawn to quantify tumor to background ratio. B, Graph bar showing fluorescence intensity of the tumor and the background at 24, 48, 72, and 96 hours post conjugates injection. C, Tumor-to-background ratio at 24, 48, 72, and 96 hours after the injection of the conjugates. Error bars represent the SE across individuals. D,Ex vivo fluorescence images of the resected organs. The white dotted lines delineate the regions used to quantify the fluorescence signals. E, MFI for each organ at each time point and for the background card (BG). Fluorescence intensity decreases over time, with the anti–GD2-IR800 being consistently brighter than the anti–GD2-IR12 (MFIIR800/MFIIR12 = 1.90 ± 0.36 for tumor, P = 10−20, three-way ANOVA). Error bars represent the SE across individuals. F, MFI of tumor relative to MFI of each organ at each time point. Data for control mice is shown in green in the graphs B, C, E, and F.

Supplementary Figures 1-6 from Proapoptotic Kinase MST2 Coordinates Signaling Crosstalk between RASSF1A, Raf-1, and Akt

Overview of the in vivo preclinical study. A, Six to 8-week-old athymic nude female mice LAN-1 xenografts were injected with either anti–GD2-IR800 or anti–GD-IR12. The control mice were not injected. Mice were imaged at 24, 48, 72, and 96 hours using the IVIS Spectrum and the multispectral NIR-I/SWIR fluorescence imaging system. B, At each end time point, mice were euthanized and imaged using the IVIS (n = 3) and the multispectral NIR-I/SWIR fluorescence imaging system (n = 1). The unexposed tumors were imaged, followed by the exposed tumors and the excised organs of interest.

Imaging of stained cell pellets beneath tissue-mimicking material using multispectral NIR-I/SWIR fluorescence imaging. A, Microcentrifuges tubes containing pellets of 2 × 106 GD2-positive cells (LAN-1) and GD2-negative cells (SUPT1-WT) stained with 100 nmol/L of anti–GD2-IR800 and covered with a 2% emulsion intralipid. The pellet is located at the tip of the tube. The fluid level is marked in red on the Petri dish. B and C, Multispectral NIR-I/SWIR fluorescence images were captured. Images show the anti–GD2-IR800–stained GD2-positive cells (LAN-1; B) and the anti–GD2-IR800–stained GD2-negative cells (SUPT1-WT; C). White dotted lines show the approximate location of the microcentrifuge tube. D, Line profiles across the fluorescence images were fitted with a Gaussian plus linear background to extract a height and FWHM. Fits shown as a black line. E–G, Bar graphs of the height (E and F) and the FWHM (G and H) versus depth for each wavelength band for GD2-positive (LAN-1) cells stained with anti–GD2-IR800 (E and G) and anti–GD2-IR12 (F and H).

Supplementary Figure from HER2 Mediates PSMA/mGluR1-Driven Resistance to the DS-7423 Dual PI3K/mTOR Inhibitor in PTEN Wild-type Prostate Cancer Models

SWIR imaging achieves a higher TBR than NIR-I imaging. A, Normalized band images of exposed tumors at 24 hours post conjugates injection. Images show increased TBR at longer wavelengths. B, Normalized band images (900 and 1,300 nm only) 48, 72, and 96 hours after anti–GD2-IR800 and anti–GD2-IR12 administration. C, TBR versus wavelength band for each conjugate and control mice. Each line represents an individual (one individual per dye per time point). The TBR measured at 1,300 nm 72 hours post anti–GD2-IR12 injection was omitted as an outlier. The upper black line shows the fit of the combined anti–GD2-IR800 and anti–GD2-IR12 data. The lower black line shows the fit of the control data. The gray bands show the 95% confidence interval of the fit (simultaneous functional bounds).