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

Research ArticleImmunologyOncology Open Access | 10.1172/JCI162139

Winson S. Ho,1 Isha Mondal,1 Beisi Xu,2 Oishika Das,1 Raymond Sun,1 Pochin Chiou,1 Xiaomin Cai,3 Foozhan Tahmasebinia,4 Caren Yu-Ju Wu,5 Zhihao Wu,4 William Matsui,6 Michael Lim,5 Zhipeng Meng,3 and Rongze Olivia Lu1,7

1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

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1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

Find articles by Wu, C. in: JCI | PubMed | Google Scholar

1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

Find articles by Wu, Z. in: JCI | PubMed | Google Scholar |

1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

Find articles by Matsui, W. in: JCI | PubMed | Google Scholar

1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

Find articles by Lim, M. in: JCI | PubMed | Google Scholar

1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

Find articles by Meng, Z. in: JCI | PubMed | Google Scholar

1Department of Neurosurgery, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

2Center for Applied Bioinformatics, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

3Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, USA.

4Department of Biological Sciences, Southern Methodist University, Dallas, Texas, USA.

5Department of Neurosurgery, Stanford University, Stanford, California, USA.

6Department of Oncology, Dell Medical School, The University of Texas at Austin, Austin, Texas, USA.

7Helen Diller Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

Address correspondence to: Rongze Olivia Lu, 1450 3rd St, Room 482, San Francisco, California 94158, USA. Phone: 626.353.3892; Email: rongze.lu@ucsf.edu. WSH, IM, OD, and ROL’s present address is: Department of Neurological Surgery, University of California, San Francisco, California, USA.

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

Find articles by Lu, R. in: JCI | PubMed | Google Scholar

Authorship note: WSH, IM, BX, and OD contributed equally to this work.

Published February 9, 2023 - More info

Published in Volume 133, Issue 6 on March 15, 2023
J Clin Invest. 2023;133(6):e162139. https://doi.org/10.1172/JCI162139.
© 2023 Ho et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published February 9, 2023 - Version history
Received: May 25, 2022; Accepted: February 2, 2023 View PDF Abstract

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.

Graphical Abstractgraphical abstract Introduction

Cyclic GMP–AMP synthase/stimulator of IFN genes (cGAS/STING) is a critical sensor for cytosolic double stranded DNA (dsDNA) to elicit antitumor immunity (14). cGAS binding to dsDNA leads to the formation of 2′,3′-cyclic GMP–AMP (cGAMP) that activates STING and induces the phosphorylation of IFN regulatory factor 3 (IRF3) to promote Type I IFN production and antitumor immune responses (35). Both tumor cells and myeloid cells express cGAS/STING, but accumulating evidence suggests that cGAMP is primarily produced by tumor cells and released as an immunotransmitter to activate STING in myeloid cells and stimulate antitumor immunity by triggering Type I IFN production (68). Despite promising preclinical studies, several small-molecule agonists of STING have shown limited clinical efficacy in early phase clinical trials (9). It is possible that these agonists cannot fully activate and sustain STING-Type I IFN signaling because the mechanisms that normally attenuate STING signaling remain engaged in the tumor microenvironment. The steps involved in STING activation are well characterized, but the mechanisms that serve to downregulate STING are not well understood. As specific phosphorylation events are required for STING activation, it is likely that dephosphorylation is involved in attenuating signaling.

Protein phosphatase 2A (PP2A) is a major protein phosphatase that accounts for 50%–70% of the total serine/threonine phosphatase activity in eukaryotic cells to counterbalance the regulatory effects of kinases in modulating numerous signaling pathways (1012). PP2A consists of various subunits, such as regulatory, B; scaffolding, A; and catalytic,C subunits. Different combinations of A, B and C subunits can lead to 60 different PP2A holoenzymes with distinct functions in different cell types (11, 12). The specificity of PP2A holoenzymes is determined by a heterogenous family of regulatory B subunits. Our group was the first to report that pharmacological inhibition of the PP2A catalytic subunit C (PP2Ac) enhances the efficacy of immune checkpoint blockade in multiple PD-1-resistant mouse tumor models (13, 14). However, given the ubiquity of PP2A expression in many cell types and the promiscuity of PP2A involvement in many cellular pathways, the mechanisms of how PP2A regulates antitumor immunity are unclear. Tumor-associated macrophages (TAMs) are the predominant myeloid cells in the tumor environment and are associated with poor prognosis in cancer. TAMs can promote immunosuppression and inhibit antitumor T cell responses, thereby limiting the efficacy of checkpoint inhibitors (1518). Previous studies have demonstrated that PP2A plays a critical role in regulating TLR-mediated Type-I IFN and NFkB signaling in macrophage response to viral infections (19, 20). However, the role of PP2A in TAMs, and, in particular, STING-mediated Type I IFN signaling is unexplored.

In this study, we present biochemical, genetic, and functional evidence that PP2A with its specific regulatory B subunit, STRN4, negatively regulates STING–Type I IFN signaling in macrophages. Mice with PP2Ac deficiency in macrophages exhibited reduced tumor growth, increased numbers of tumor-infiltrating CD8+ T cells, and reduced numbers of immunosuppressive macrophages. Macrophage PP2Ac deficiency also synergizes with STING agonists, radiation, and checkpoint blockade in multiple syngeneic tumor models. Single-cell RNA-Seq (scRNA-Seq) demonstrated that macrophage-specific loss of PP2Ac resulted in complex remodeling of the immune landscape with enhanced Type I IFN signature in TAMs and an increased adaptive immune response. STRN4 has been implicated in biochemical studies to regulate the Hippo-Yes-associated protein (Hippo-YAP) pathway (21), which has an established role in tumorigenesis. However, the function of STRN4 has not been described in immune cells and the role of Hippo-YAP pathways has not been explored in TAMs. We found that the Hippo kinase mammalian STE20-like protein kinase (MST1/2), a negative regulator of YAP and transcriptional coactivator with PDZ-binding motif (TAZ), is required for STING activation. Mechanistically, STRN4 associates with PP2Ac to dephosphorylate Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. We also found that tumor significantly upregulated YAP/TAZ expression in TAMs, resulting in suppression of Type I IFN signaling in the context of cGAS-STING stimulation. Thus, PP2A/STRN4-Hippo-YAP/TAZ signaling is critical in regulating STING-Type I IFN in TAMs. Our work provides the rationale for targeting this pathway to enhance antitumor immunity by combination with other STING-activating strategies.

Results

PP2Ac negatively regulates STING-Type I IFN signaling pathway. To address the effect of PP2Ac on STING signaling in macrophages, we chose the LysMcrePP2Acfl/fl mice. These mice carry floxP sites that flank exon 1 of ppp2ca, have cre expression under the Lysozyme 2 (LysM) promoter, and have a myeloid lineage, specifically the macrophages, that have a deficiency in PP2Ac (22). We generated bone marrow-derived macrophages (BMDM) from LysMcrePP2Acfl/fl and WT mice and treated them with the STING agonist cGAMP. RNA-Seq was then performed to identify global gene expression–profile changes. Pathway enrichment analysis demonstrated that IFN and TNF signaling pathways were among the highest enriched differentially expressed gene sets between PP2AcKO and PP2AcWT BMDM in response to cGAMP (Figure 1A). Since both IFN and TNF are known downstream signaling of STING activation, this result is consistent with PP2Ac-deficiency mediated STING activation. Gene set enrichment analysis (GSEA) confirmed the upregulation of gene signatures associated with Type I IFN (Figure 1B) and TNF (Figure 1C) responses in PP2AcKO compared with PP2AcWT BMDM following cGAMP stimulation. The implication of Type II (γ) IFN signaling in pathway enrichment analysis is likely due to overlapping Type I and Type II IFN gene sets and not due to IFN-γ receptor (IFNGR) activation. Indeed, BMDMs were stimulated with cGAMP in isolation without other in vitro sources of Type II IFN, which is primarily secreted by lymphocytes rather than macrophages. Therefore, the implication of Type II (γ) IFN signaling in pathway enrichment analysis is likely due to overlapping Type I and Type II IFN gene sets and not due to IFN-γ–receptor (IFNGR) activation. Next, we confirmed using RT-PCR that the expression of IFNβ and 3 critical IFN-stimulated genes (CXCL10, CXCL9, and ISG15) (23) were upregulated in cGAMP-stimulated PP2AcKO BMDM (Figure 1D). Furthermore, we examined the time course of p-IRF3 and p-STAT1 protein expression following cGAMP treatment. p-IRF3 is the downstream mediator of STING activation leading to transcription of IFNα/β, and p-STAT1 is activated by the IFNα/β receptor IFNAR, by autocrine Type I IFN stimulation. Compared with PP2AcWT, PP2AcKO BMDM had amplified activation of both pIRF3 and pSTAT1 following cGAMP stimulation. Response peaked at 6 hours, but the signal remained elevated in PP2AcKO compared with PP2AcWT at 18 hours after stimulation (Figure 1E). Production of IFNβ and TNF cytokines in the culture supernatant in response to STING stimulation was enhanced in PP2AcKO compared with PP2AcWT BMDM (Figure 1F). We also asked if PP2AcKO in BMDM enhanced antigen presenting phenotype in classically activated M1 condition by measuring the expression of MHCII, CD80, and CD86 (Figure 1G and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI162139DS1), which are essential for macrophage activation and antigen presentation. PP2AcKO BMDM treated with STING agonists had increased CD86 expression (Supplemental Figure 1B) compared with the control. We also tested the effect of pharmacologic PP2Ac inhibition using a small molecule inhibitor, LB-100. p-IRF3 (Figure 1H) and IFNβ cytokine production (Figure 1I) in mouse macrophage RAW cells were enhanced with STING agonist stimulation. To generalize our findings in human macrophages, we also generated PP2AcKO human ThP-1 cell lines using CRISPR/Cas9 gene-KO technique and confirmed that PP2Ac protein expression was absent in these cells (Supplemental Figure 2A). THP-1 cells were differentiated into macrophages by phorbol myristate acetate (PMA) for 24 hours before stimulated with STING agonists for 4 hours. Expression of IFNβ and IFN-stimulated genes were upregulated in PP2AcKO THP-1 differentiated macrophages compared with control cells (Supplemental Figure 3A). Protein expression of pIRF3 was also enhanced in PP2AcKO compared with control cells following cGAMP treatment (Supplemental Figure 3B). We further tested the effect of LB-100 on primary human macrophages. Human PBMC–derived monocytes were treated with M-CSF for 6 days to induce macrophage differentiation. Cells were then treated with LB-100 for 2 hours prior to stimulation with cGAMP. After 4 hours, expressions of IFN-stimulated genes CXCL10, CXCL9, and ISG15 were found to be significantly enhanced in LB-100–treated macrophages (Figure 1J). Cumulatively, these results demonstrated that genetic or pharmacological inhibition of PP2Ac consistently enhanced STING-Type I IFN signaling in human and murine macrophage cells.

PP2A negatively regulates STING-Type I IFN signaling pathway.Figure 1

PP2A negatively regulates STING-Type I IFN signaling pathway. (A) Pathway enrichment analysis of RNA-Seq of PP2AcKO and PP2AcWT BMDM treated with cGAMP (10 μg/mL) for 4 hours (n = 3 per group) showing the top 5 enriched pathways ranked with highest –log10 P value using differentially upregulated genes in PP2AcKO compared with PP2AcWT BMDM (Log2 fold change (log2FC) > 1, FDR < 0.01). * indicates the P value for each individual pathway. (B and C) GSEA plots for Type I IFN (B) and TNF (C) signatures between cGAMP-treated PP2AcKO versus PP2AcWT BMDM. (D) BMDM were harvested 4 hours after cGAMP stimulation (10 μg/mL). Expression of IFNβ and IFN response genes (CXCL10, CXCL9, and ISG15) were measured via reverse transcription PCR. (E) Protein expression of BMDM was analyzed by immunoblotting after cGAMP (10 μg/mL) treatment. (F) PP2AcKO and PP2AcWT BMDM were stimulated with DMXAA (10 μg/mL) for 48 hours, cytokine concentrations were measured in culture supernatant. (G) PP2AcKO and PP2AcWT BMDM were treated with IFNγ (10 ng/mL) for 24 hours, expressions of CD80, CD86, and MHCII were measured by FACS. Representative FACS plot of MHCII expression ± IFNγ treatment. (H) RAW cells were pretreated with the PP2A inhibitor LB-100 for 2 hours before stimulated with cGAMP (10 μg/mL) for 4 hours. Protein expression was analyzed by immunoblotting. (I) RAW cells were pretreated with LB-100 for 2 hours before stimulated with DMXAA (10 μg/mL) for 48 hours. Cytokine concentrations were measured in culture supernatant. (J) PBMCs were treated with M-CSF (50 ng/mL) for 6 days to derive macrophages. Cells were then pretreated with LB-100 at the indicated dosage for 1.5 hours prior to cGAMP (10 μg/mL) treatment. Expression of IFN response genes (CXCL10, CXCL9, and ISG15) were measured via real time PCR. Data are from 1 experiment representative of at least 2 (BI) and 1 (J) independent experiments with similar results. Error bars depict SEM. P values were calculated by unpaired 2-tailed t test *P < 0.05,**P < 0.01, ***P < 0.001, ****P < 0.0001.

Deficiency of PP2Ac in macrophages reduces tumor growth and alters tumor immune microenvironment. To test our hypothesis that PP2AcKO in macrophages can enhance antitumor immunity, we implanted B16 melanoma, SB28 glioma, and MC38 colon tumor cells s.c. in LysMcrePP2Acfl/fl and WT mice. We chose these cell lines because of their variable range of intrinsic immunogenicity. Tumor growth was all significantly reduced in LysMcrePP2Acfl/fl mice (Figure 2, A–C), suggesting that macrophage-specific PP2Ac deficiency can induce a potent antitumor effect. We then assessed the functional consequence of macrophage PP2Ac deletion on tumor-infiltrating leukocytes (TILs) and tumor-draining lymph node-resident (tumor-dLN-resident) T cells in B16 melanoma. Ten days after implantation, tumors and tumor-dLN were harvested and analyzed by flow cytometry. We found increased infiltration of CD8+ and CD4+ T cells in the tumor (Figure 2D and Supplemental Figure 4A) and systemically in the spleen (Supplemental Figure 4B). We did not find a significant change in total F4/80 macrophage tumor infiltration (Supplemental Figure 4C). However, consistent with our in vitro findings, we found significantly enhanced expression of MHCII in F4/80+ macrophages (Figure 2E), suggesting an enhanced proinflammatory phenotype in tumor-infiltrating macrophages. In addition, the frequency of immunosuppressive polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) was significantly decreased (Figure 2F). In tumor-dLN, there was an increased frequency of resident CD4+ T cells (Supplemental Figure 4E) and enhanced IFNγ and IFNγ+ TNF producing CD8+ and CD4+ T cells in mice with macrophage-specific PP2AcKO (Figure 2, G and H, and Supplemental Figure 4F). To confirm the generalizability of the immunomodulatory effect of macrophage-specific PP2AKO on TILs in other cancer models, we also examined orthotopic intracranial (i.c.) GL261 glioma. The brain tumor microenvironment is known to be more immunosuppressive compared with s.c. tumors (24). We similarly found increased CD8+ and CD4+ T cell infiltration (Supplemental Figure 5, A and B). The frequency of monocytic derived macrophages (CD11b+CD49d+) — a subpopulation of TAMs in brain tumors known to correlate with the clinical outcome (18) — was significantly decreased in LysMcrePP2Acfl/fl mice (Supplemental Figure 5C). TAMs were found to have increased expression of activation markers MHCII and CD86 and decreased immunosuppressive marker CD206 (25, 26) in LysMcrePP2Acfl/fl mice (Supplemental Figure 5D). The fact that we observed increased tumor infiltration of cytotoxic T cells suggested that macrophage PP2Ac deficiency can elicit enhanced adaptive antitumor immunity. We asked whether the increased CD8+ T cell response was required for the antitumor effect in LysMcrePP2Acfl/fl mice. We performed systemic CD8+ T cell depletion by treating WT and LysMcrePP2Acfl/fl mice with isotype control or anti-CD8 depleting antibody prior to tumor implantation and throughout the study. CD8 depletion completely abolished the benefit of LysMcrePP2Acfl/fl mice, suggesting that CD8-mediated adaptive immune response was required for the beneficial effects of macrophage PP2Ac KO (Figure 2I). In summary, these data suggest that macrophage PP2Ac–deficiency enhanced T cell effector function by remodeling the myeloid compartment of the tumor immune microenvironment.

Macrophage PP2Ac deficiency reduces tumor growth and alters the tumor immunFigure 2

Macrophage PP2Ac deficiency reduces tumor growth and alters the tumor immune microenvironment. (AC) LysMcrePP2Acfl/fl or WT C57BL/6 mice were inoculated with 0.1 × 106 (A) B16, (B) SB28, or (C) 1 × 106 MC38 cells s.c. (n = 8–10). (DG) B16 tumors were implanted s.c. in LysMcrePP2Acfl/fl or WT mice. Mice were euthanized on day 10. Tumors were harvested for tumor infiltrating leukocyte (TIL) profiling (DF) and tumor-draining lymph node (tumor-dLN) (G and H) by flow cytometry (n = 9–10). (D) Quantification of CD4+ and CD8+ TILs and representative FACS plot. (E) Quantification of MHCII+ expression in tumor infiltrating macrophages (F4/80+) with representative FACS plot. (F) Quantification of Ly6G+Ly6Clo PMN-MDSC in TILs. (GH) Quantitation of IFNγ-producing or IFNγ/TNF dual–producing dLN-resident CD8+ (G) and CD4+ (H) T cells as percentages of total CD8+ and CD4+ T cells, respectively. IFNγ and/or TNF production was stimulated exvivo with PMA/ionomycin in conjunction with protein transport inhibitor for 4 hours prior to staining. Representative FACS plots of dLN CD8+ T cells after stimulation. (I) LysMcrePP2Acfl/fl or WT C57BL/6 mice were treated with anti-CD8 depletion antibody or isotype control. Mice were given 250 μg i.p. on day –3, –2, and –1, then inoculated with 0.1 × 106 B16 cells s.c. (day 0) (n = 8). Depleting antibody or isotype was then given 2 × per week until endpoint. Data are from 1 experiment representative of at least 2 (AH) and 1 (I) independent experiments with similar results. Error bars depict SEM. P values were calculated by unpaired 2-tailed t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Deficiency of PP2Ac in macrophages sensitizes tumors to STING agonists, radiation, and immune checkpoint blockade. Given our in vitro finding that PP2Ac-deficient macrophages have enhanced Type I IFN signaling (Figure 1A), we asked whether type I IFNs was required for the antitumor effect of macrophage PP2Ac KO in vivo. Mice bearing SB28 (Figure 3A) or B16 (Supplemental Figure 6A) tumors were treated with intratumoral injections of isotype or IFNAR-blocking antibody on days 0 and 2 after tumor implantation followed by biweekly injections in LysMcrePP2Acfl/fl or WT mice. Blocking type I IFN signaling abrogated the therapeutic effect of macrophage specific PP2Ac deficiency in both models and restored tumor sizes to levels similar to WT mice, suggesting that type I IFNs are essential for eliciting PP2Ac-deficiency–mediated antitumor response in TAMs. We then asked whether blockade of type I IFN signaling will affect the degree of CD8+ T cell infiltration in the tumor microenvironment. At survival endpoint, tumors were harvested and stained for CD8+ T cells by immunofluorescence. We found that the increase in tumor infiltrating CD8+ T cells in LysMcrePP2Acfl/fl mice was significantly reduced once IFN signaling was blocked (Figure 3B), suggesting that Type I IFN signaling was required for PP2Ac-deficient macrophages to promote enhanced adaptive antitumor immunity.

Macrophage PP2Ac deficiency synergizes with STING agonist, radiation, and iFigure 3

Macrophage PP2Ac deficiency synergizes with STING agonist, radiation, and immune checkpoint blockade. (A) LysMcrePP2Acfl/fl or WT C57BL/6 mice were inoculated with 0.1 × 106 SB28 cells s.c. (n = 8). Mice were given intra-tumoral injection of anti-IFNAR-1 or isotype control (100 μg) on days 0 and 2, and then 2 times per week. (B) At survival endpoint, histological analysis was performed, staining for CD8 (red) and nucleus (4,6-diamidino-2-phenylindole (DAPI), blue). Scale bar: 10 μm. CD8 cells per field of view from 3 areas of interest on 3 independent samples (n = 9) were quantified. (C) LysMcrePP2Acfl/fl or WT mice were inoculated with 0.1 × 106 SB28 cGASKO (n = 8) cells s.c. (D) LysMcrePP2Acfl/fl or WT mice were inoculated with 0.1 × 106 B16 cells s.c. At day 4, mice were randomized (n = 7-8) into intratumoral injection of PBS or cGAMP (3 μg) at days 4, 8, and 11. (E) LysMcrePP2Acfl/fl or WT mice were inoculated with 0.1 × 106 B16 cells s.c. At day 7, mice were randomized to with or without radiation (n = 8). For the radiation groups, tumors were locally irradiated with 3Gy daily for 3 consecutive days (3 × 3Gy). (F) LysMcrePP2Acfl/fl or WT mice were inoculated with 3 × 104 SB28 cells in the brain. At day 5, mice were randomized to with or without radiation (n = 9–10). Cumulative survival of mice over time. (G) LysMcrePP2Acfl/fl or WT mice were inoculated with 1 × 106 MC38 cells s.c. At day 7, animals were randomized to treatment with anti-PD-1 or IgG1 isotype (200 μg) antibodies via i.p injection, given twice a week. Data are from 1 experiment representative of at least 2 (for CG) or 1 (for A and B) independent experiments with similar results. Mantel-Cox log-rank tests were used for survival analysis. Error bars depict SEM. P values were calculated by 1-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Since we found that macrophage PP2Ac deficiency enhanced STING-mediated IFN signaling (Figure 1, D–J) in vitro, we asked if promotion of STING activation in macrophages was responsible for enhanced antitumor immunity in LysMcrePP2Acfl/fl mice in vivo. Several recent studies demonstrated that the export of tumor-derived cGAMP to activate STING in host immune cells is essential to eliciting a successful antitumor response (6, 8, 27). We tested if cGAS KO in tumor cells, which will deplete the source of cGAMP to activate host STING, will abolish the therapeutic effect of macrophage PP2Ac deficiency. To this end, we generated cGASKO in B16 and SB28 cell lines using CRISPR/Cas9 and confirmed that cGAS protein expression was absent in these cells (Supplemental Figure 2, B and C). We found that the effect of reduced tumor growth in LysMcrePP2Acfl/fl mice was abolished when cGAS was deficient in SB28 (Figure 3C) or B16 (Supplemental Figure 6B) tumors, suggesting that tumor cGAMP production, which is responsible for STING activation in host macrophages, is required for PP2Ac-mediated regulation of macrophage tumor immunity.

Next, since administration of STING ligands has bee

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