PP2Ac/STRN4 negatively regulates STING-type I IFN signaling in tumor-associated macrophages

Stimulator of IFN genes type I (STING-Type I) IFN signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonists as monotherapy have shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in macrophages. Mice with macrophage PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human patients with glioblastoma (GBM), YAP/TAZ was highly expressed in tumor-associated macrophages but not in nontumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor-conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in tumor-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy.

with a blocking buffer consisting of 5% normal goat serum and 0.3% Triton in PBS for one hour at room temperature. Primary antibodies were then added at 1:200 dilutions of mouse anti-human YAP/TAZ antibodies (Santa Cruz, 101199) and incubated at 4 °C overnight. The cells were then washed and incubated with 1:200 dilutions of AF4888-labeled secondary goat anti-mouse antibodies (Abcam 150113) or Alex594-labelled secondary goat anti-rabbit antibodies (Abcam 150120), and then mounted onto glass slides using vectaSHIELD HardSet, Antifade mounting medium with DAPI. (Vector Laboratories H-1500). Confocal fluorescence images were collected using a ×40 oil-immersion objective (Nikon A1R confocal).

Quantification of Immunofluorescence staining
For quantification of YAP signaling from immunofluorescence imaging (in Figure 7B and Supplemental Figure 16A), the mean pixel intensity of the YAP channel was collected using the measure tool in Fiji (ImageJ). Acquisition parameters, such as lens objective, pinhole settings, and laser power, were kept consent. Five areas of 1024 X 1024 pixels 2 were randomly selected and imaged per sample. To adjust the background signal, the LUTs were changed equally among all images, then the composite images were split based on the color channel and the threshold was adjusted on the YAP staining channel to set the analysis region of interest (ROI). The total YAP intensity of each ROI was measured and normalized by the total number of pixels, adjusting for the pixel count variation within the region of interest among each of the five images. The mean pixel intensities were then averaged for each of the five regions.

In vivo immune cell profiling
For intratumoural T cells and T-cell effector cytokine expression, single-cell suspensions were prepared from fresh tumor tissues using the Tumor Dissociation Kit (Miltenyi, with the gentleMACS Octo Dissociator (Miltenyi) per manufacture instruction. For draining lymph nodes, tissues were dissociated in a 70 µm mesh and washed several times with FACS buffer to obtain single cell suspension. Fc receptors were blocked with TruStain FcX anti-mouse CD16/32 (BioLegend, 101319) in 20 µg/mL for 10 mins at 4°C. Cells were stained with surface markers in 4°C for 30 mins.
For cytokine staining, cells were incubated with Cell Stimulation Cocktail (Thermo Scientific, in RPMI medium containing PMA ionomycin, brefeldin A and monensin at 37 °C for 4 h before undergoing surface staining. The cells were then washed and resuspended in IC Fixation Buffer (Thermo Scientific, 00-8222) at room temperature for 20 mins then wash with Perm/Wash buffer (Thermo Scientific, 00-8333). Cells were then stained with specific intracellular markers. All the samples were read using the Cytek Aurora cytometer and analyzed using SpectroFlo (Cytek Bioscience) and FlowJo software.

Survival analysis
Normalized expression levels for GBM, LGG, SKCM and BRCA were downloaded from Broad GDAC Firehose portal (level 3 RSEM) (Broad). For each patient data, mean expression levels of the marker genes of the cluster of interest were calculated. Patients were then grouped into 4 quartiles that Q1 were expressed lower level of marker genes. Then survival analysis was performed at cBioportal in Merged Cohort of LGG and GBM(52), SKCM and BRCA(53, 54).

RNA sequencing of BMDM
BMDM from WT and PP2Ac were generated as described above. They were them treated with STING agonist DMAXX (InvivoGen), 10 ug/ml, for 4h before RNA extraction. Total RNA was isolated using the PureLink™ RNA Mini Kit (Invitrogen, 12183025). RNA sequencing was performed by the University of Texas Genomic Sequencing and Analysis Facility. Poly(A) enrichement was performed. mRNA was isolated from total RNA using the Poly(A) Purist-MAG kit (ThermoFisher). Briefly, total RNA was combined in a 96-well plate with washed and resuspended Oligo (dT) MagBeads. The mixture was heated in a thermocycler set to 70°C for 5 minutes, followed by incubation at room temperature with gentle vortexing for 30 minutes. Subsequently, the beads were captured and washed, and bound poly(A) RNA was eluted in water. mRNA quality was assessed on the Agilent Bioanalyzer using the Agilent RNA 6000 Pico kit (Agilent). Libraries were prepared at the University of Texas Genomic Sequencing and Analysis Facility according to manufacturer's instructions for the NEBNext Ultra II Directional RNA kit (NEB, product number E7760). The resulting libraries tagged with unique dual indices were checked for size and quality using the Agilent High Sensitivity DNA Kit (Agilent). Library concentrations were measured using the KAPA SYBR Fast qPCR kit and loaded for sequencing on the NovaSeq 6000 instrument (paired end 2X150). Reads were first trimmed for adapters using fastp raw counts were downloaded from Brain Time portal(45, 47) and differential analysis were performed using Voom like mentioned above.

Mouse scRNASeq analysis
SB28 tumors were inoculated in the flank, or the brain, as described above, in PP2Ac WT or PP2Ac KO mice. 18 days after implantation, tumor tissue was harvested, and single cell suspensions were obtained using Tumor Dissociation Kit (Miltenyi,