MAO-A-deficient mice show reduced tumour growth associated with altered TAM polarization
In a search for new molecules regulating TAM reprogramming, we inoculated C57BL/6J mice with syngeneic B16-OVA melanoma tumours, isolated TAMs, and assessed TAM gene expression profiles. Monocytes isolated from tumour-free and tumour-bearing mice were included as controls. In addition to changes in classical genes involved in regulating macrophage immune responses, we observed the induction of a Maoa gene in TAMs (Fig. 1a), suggesting that MAO-A may be involved in modulating TAM activities.
To study the role of MAO-A in antitumour immunity in vivo, we used MAO-A-deficient mice that carry a hypomorphic MAO-A mutant 56. Although a degree of Maoa expression leakage in the brain had been previously reported in these mice 56, analysis of their immune system showed a nearly complete ablation of MAO-A expression in major lymphoid organs including spleen and bone marrow (BM) (Supplementary Fig. 1a). Since we focused on immune cells in this study, we denote these mice as Maoa knockout (KO) mice. When challenged with B16-OVA melanoma cells (Fig. 1b), tumour growth in Maoa KO mice was significantly suppressed compared to that in Maoa wildtype (WT) mice (Fig. 1c,d). Although similar levels of TAMs (gated as CD45.2+CD11b+Ly6G-Ly6C-/lowF4/80+ cells) were detected in Maoa WT and Maoa KO mice (Supplementary Fig. 1b,c), compared to their WT counterparts, TAMs isolated from Maoa KO mice exhibited a less immunosuppressive phenotype, indicated by their decreased expression of immunosuppressive markers (i.e., CD206; Fig. 1e), and their increased expression of immunostimulatory molecules (i.e., CD69, CD86, and MHC class II I-Ab; Fig. 1f-h). Further analysis showed that TAMs from Maoa KO mice expressed reduced levels of immunosuppression- associated genes (i.e., Mrc1, Chi3l3, and Arg1; Fig. 1i) and increased levels of pro-inflammatory cytokine genes (i.e., Il6, Tnfα, and Ccl2; Fig. 1j). Corresponding to the altered TAM polarization in Maoa KO mice, tumour-infiltrating CD8+ T cells in these mice showed enhanced activation (i.e., increased production of Granzyme B; Supplementary Fig. 1d). Single-cell RNA sequencing (scRNAseq) analysis was performed on tumour infiltrating immune cells isolated from Maoa WT and Maoa KO mice (Fig. 1k and Supplementary Fig. 1e,f). UMAP analysis of extracted TAMs showed a reduced immunosuppressive phenotype in Maoa KO mice, with an increased ratio of Mrc1lowCd86high cells to Mrc1highCd86low cells (Fig. 1l and Supplementary Fig. 1g). Gene expression profile analysis confirmed a reduction of TAMs expressing immunosuppressive genes (i.e., Mrc1 and Chi3I3; Fig. 1m) and an enrichment of TAMs expressing immunostimulatory genes (i.e., Ccl2, Ccl7, Cd86, H2-Aa, and H2-Ab1; Fig. 1n) in Maoa KO mice. These data strongly indicate that MAO-A is involved in regulating TAM polarization thereby modulating antitumour immunity.
MAO-A directly regulates TAM polarization and influences TAM-associated T cell antitumour reactivity
In our Maoa KO mice tumour challenge study, MAO-A deficiency impacted both immune and non-immune cells (Fig. 1b). To determine whether MAO-A directly regulates immune cells, we conducted a BM transfer experiment wherein BM cells harvested from Maoa WT or KO mice were adoptively transferred into BoyJ (CD45.1) WT recipient mice followed by B16-OVA tumour challenge (Fig. 2a). In this experiment, MAO-A deficiency comparison was confined to immune cells. MAO-A deficiency in immune cells resulted in suppressed tumour growth (Fig. 2b,c), altered TAM polarization (i.e., downregulation of immunosuppressive markers such as CD206, Fig. 2d; and upregulation of immunostimulatory markers such as CD69, CD86, and MHC class II I-Ab; Fig. 2e,f and Supplementary Fig. 2a), and enhanced tumour-infiltrating CD8+ T cell activation (i.e., increased production of cytotoxic molecules such as Granzyme B; Supplementary Fig. 2b), indicating that MAO-A directly regulates immune cell antitumour activity, in particular TAM polarization and T cell antitumour reactivity.
To further study whether MAO-A acts as a macrophage autonomous factor directly regulating TAM polarization and thereby influencing antitumour immunity, we performed a macrophage adoptive transfer tumour experiment. BM cells were harvested from Maoa WT and KO mice then cultured into bone marrow-derived macrophages (BMDMs). These Maoa WT or KO BMDMs were then mixed with B16-OVA melanoma cells and subcutaneously (s.c.) injected into BoyJ WT recipient mice to establish solid tumours (Fig. 2g). In this study, MAO-A deficiency comparison was confined to TAMs. Suppressed tumour growth (Fig. 2h,i), downregulated expression of TAM immunosuppressive markers (i.e., CD206; Fig. 2j), upregulated expression of TAM immunostimulatory markers (i.e., CD69 and CD86; Fig. 2k,l), and enhanced tumour- infiltrating CD8+ T cell reactivity (i.e., increased production of Granzyme B; Fig. 2m) were observed in mice receiving Maoa KO BMDMs. Collectively, these in vivo studies demonstrate that MAO-A acts as an autonomous factor directly regulating TAM polarization, and thereby influencing T cell antitumour reactivity and impacting tumour growth.
MAO-A promotes macrophage immunosuppressive polarization
To study MAO-A regulation of macrophage polarization, we cultured Maoa WT and KO BMDMs in vitro and polarized these macrophages toward an immunosuppressive phenotype by adding anti- inflammatory stimuli (i.e., IL-4 and IL-13; Fig. 3a). We observed a sharp induction of Maoa mRNA expression in Maoa WT BMDMs during macrophage development, that remained high post-IL-4/IL-13 stimulation (Fig. 3b,c). MAO-A expression was undetectable in Maoa KO BMDMs, confirming their Maoa-deficiency genotype (Fig. 3b,d). Compared to their wildtype counterpart, Maoa KO macrophages displayed a less immunosuppressive phenotype under IL- 4/IL-13 stimulation, evidenced in their reduced expression of immunosuppressive markers (i.e., CD206; Fig. 3e) and signature genes (i.e., Chi3l3 and Arg1; Fig. 3f,g). When tested in a macrophage/T cell co-culture assay (Fig. 3h), in agreement with their less immunosuppressive phenotype, IL-4/IL-13-polarized Maoa KO macrophages exhibited impaired suppression of wildtype CD8+ T cells under anti-CD3/CD28 stimulation, shown as their attenuated inhibition of CD8+ T cell proliferation (Fig. 3i) and activation marker expression (i.e., upregulation of CD25 and CD44, and downregulation of CD62L; Fig. 3j,k and Supplementary Fig. 3a).
To verify whether MAO-A deficiency directly contributed to the alleviated immunosuppressive polarization of Maoa KO macrophages, we performed a rescue experiment. We constructed a MIG-Maoa retroviral vector, used this vector to transduce Maoa KO BMDMs, and achieved overexpression of MAO-A in these macrophages (Fig. 3l-n, and Supplementary Fig. 3b). MAO-A overexpression significantly exacerbated the immunosuppressive phenotype of IL- 4/IL-13-stimulated Maoa KO BMDMs (i.e., upregulation of immunosuppressive signature genes such as Chi3l3 and Arg1; Fig. 3o,p). Taken together, these results indicate that MAO-A acts as an autonomous factor promoting macrophage immunosuppressive polarization under anti- inflammatory stimuli.
MAO-A promotes macrophage immunosuppressive polarization via ROS upregulation
Next, we sought to investigate the molecular mechanisms regulating MAO-A promotion of macrophage immunosuppressive polarization. It has been reported that intracellular reactive oxygen species (ROS; hence, oxidative stress) elicit macrophage immunosuppressive features 57, 58, 59, 60, 61. MAO-A catalyzes the oxidative deamination of monoamines, thereby generating hydrogen peroxide (H2O2) as a byproduct that can increase intracellular ROS levels. We therefore speculated that MAO-A might promote TAM immunosuppressive polarization in TME via upregulating ROS levels in TAMs (Fig. 4a).
To test this hypothesis, we directly measured ROS levels in TAMs isolated from Maoa WT and KO mice bearing B16-OVA tumours and detected significantly lower levels of ROS in Maoa KO TAMs (Fig. 4b,c). Measurement of ROS levels in in vitro-cultured Maoa WT and KO BMDMs also showed reduced levels of ROS in Maoa KO BMDMs, with or without IL-4/IL-13 stimulation, in agreement with the in vivo TAM results (Fig. 4d). Supplementing H2O2 to IL-4/IL-13-stimulated Maoa WT and KO BMDMs elevated their intracellular ROS to similar levels (Supplementary Fig. 4a,b) and eliminated their differences in expression of immunosuppressive markers (i.e., CD206; Fig. 4e) and signature genes (i.e., Chi3l3 and Arg1; Fig. 4f,g).
On the other hand, supplementation of tyramine, a substrate of MAO-A, increased ROS levels and upregulated the expression of immunosuppressive genes (i.e., Chi3l3 and Arg1) in Maoa WT BMDMs but not in Maoa KO BMDMs (Fig. 4h-j). Taken together, these data indicate that MAO-A regulates macrophage immunosuppressive polarization via modulating macrophage intracellular ROS levels.
The JAK-Stat6 signaling pathway plays a key role in mediating IL-4/IL-13-induced immunosuppressive polarization of TAMs in TME 62, 63. After IL-4/IL-13 stimulation, JAK is phosphorylated and subsequently phosphorylates Stat6; phosphorylated Stat6 dimerizes and migrates to the nucleus, where it binds to the promoters of IL-4 and IL-13 responsive genes including those involved in macrophage immunosuppressive functions 64, 65. ROS has been reported to promote JAK and Stat6 phosphorylation in a variety of cell types 61, 66, 67, 68, 69, 70, 71. Since we observed decreased ROS levels in Maoa KO macrophages compared to those in Maoa WT macrophages (Fig. 4b,c), we postulated that MAO-A may impact macrophage polarization through upregulating ROS levels and thereby sensitizing the JAK-Stat6 signaling pathway. Indeed, direct analysis of TAMs isolated from B16-OVA tumour-bearing Maoa WT and Maoa KO mice confirmed that compared to wildtype TAMs, MAO-A-deficient TAMs showed reduced Stat6 activation (i.e., reduced Stat6 phosphorylation; Fig. 4k,l). Further analysis of IL-4/IL-13-induced JAK-Stat6 signaling pathway in Maoa KO BMDMs compared to that in Maoa WT BMDMs showed significantly reduced JAK-Stat6 signaling (i.e., reduced JAK1, JAK2, JAK3, and Stat6 phosphorylation; Fig. 4m). Supplementing H2O2 to IL-4/IL-13-stimulated Maoa WT and KO
BMDMs increased their JAK-Stat6 signaling to similar levels (i.e., comparable JAK1, JAK2, JAK3, and Stat6 phosphorylation; Fig. 4m), corresponding to their comparable high levels of ROS (Supplementary Fig. 4a,b). These data indicate that MAO-A promotes macrophage immunosuppressive polarization via ROS-sensitized JAK-Stat6 pathway activation.
Collectively, these in vivo and in vitro data support a working model that MAO-A promotes TAM immunosuppressive polarization in TME, at least partly through upregulating TAM intracellular ROS levels and thereby enhancing the IL-4/IL-13-induced JAK-Stat6 signaling pathway.
MAO-A blockade for cancer immunotherapy- syngeneic mouse tumour model studies
The identification of MAO-A as a key regulator of TAM immunosuppressive polarization makes MAO-A a promising new drug target for cancer immunotherapy. Because of the known functions of MAO-A in the brain, small molecule MAOIs have been developed and clinically utilized for treating various neurological disorders, making it a highly feasible and attractive approach to repurpose these established MAOI drugs for cancer immunotherapy 51, 72. In an in vitro WT BMDM IL-4/IL-13-induced polarization culture (Fig. 5a), addition of multiple MAOIs efficiently reduced ROS levels in BMDMs (Fig. 5b) and suppressed their immunosuppressive polarization, evidenced by their decreased expression of immunosuppressive markers (i.e., CD206; Fig. 5c) and immunosuppressive genes (i.e., Chi3l3 and Arg1; Fig. 5d,e). Notably, the MAOIs that we tested include phenelzine, clorgyline, mocolobemide, and pirlindole, covering the major categories of established MAOIs classified on the basis of whether they are nonselective or selective for MAO- A, and whether their effect is reversible (Fig. 5a) 51, 54, 73. Among these MAOIs, phenelzine (trade name: Nardil) is clinically available in the United States 72. In the following studies, we chose phenelzine as a representative to study the possibility of repurposing MAOIs for cancer immunotherapy, using two syngeneic mouse tumour models: a B16-OVA melanoma model and a MC38 colon cancer model 74.
First, we studied the therapeutic potential of phenelzine in a B16-OVA tumour prevention model (Fig. 5f). Phenelzine treatment effectively suppressed B16-OVA tumour growth in B6 wildtype mice (Fig. 5g,h). No tumour growth difference was observed when we depleted TAMs in experimental mice via a clodronate liposome treatment, indicating that phenelzine suppressed tumour growth via modulating TAMs (Fig. 5g,h and Supplementary Fig. 5a). Correspondingly, TAMs isolated from phenelzine-treated mice displayed a less immunosuppressive phenotype (i.e., decreased expression of CD206; Fig. 5i) that was correlated with an enhanced antitumour reactivity of tumour-infiltrating CD8+ T cells (i.e., increased production of Granzyme B; Fig. 5j) in these mice. Further studies showed that phenelzine treatment also effectively suppressed the progression of pre-established solid tumours in both B16-OVA and MC38 models (Supplementary Fig. 5b-f).
Next, we evaluated the potential of phenelzine for combination therapy, in particular combining with other ICB therapies such as PD-1/PD-L1 blockade therapy (Fig. 5k). Although most ICB therapies target CD8+ T cells, these cells are in fact closely regulated by TAMs in the TME, making targeting TAMs another potential avenue for immunotherapy 14, 39. In both B16- OVA and MC38 tumour models, phenelzine treatment significantly suppressed the progression of pre-established solid tumours at a level comparable to the anti-PD-1 treatment; importantly, the combination of phenelzine and anti-PD-1 treatments yielded synergistic tumour suppression efficacy (Fig. 5l-o). These tumour suppression effects of phenelzine were due to immunomodulation but not direct tumour inhibition, because phenelzine treatment did not suppress the growth of B16-OVA and MC38 tumours in immunodeficient NSG mice (Supplementary Fig. 5g-k).
Collectively, these syngeneic mouse tumour model studies provided proof-of-principle evidence for the cancer immunotherapy potential of MAOIs via targeting TAM reprogramming and thereby enhancing antitumour T cell responses.
MAO-A blockade for cancer immunotherapy- human TAM and clinical data correlation studies
To explore the translational potential of MAO-A blockade therapy, we first studied MAO-A regulation of human macrophage polarization. Using a Tumour Immune Dysfunction and Exclusion (TIDE) computational method 75, we analyzed the gene expression signatures of in vitro cultured immunostimulatory M1-like and immunosuppressive M2-like human monocyte-derived macrophages (MDMs) (GSE35449) 76. Interestingly, among all immune checkpoint and immune suppressive genes examined, MAOA ranked as the top gene with the most dramatically elevated expression in M2-like MDMs (i.e., 7.28 M2/M1 log-fold change; Fig. 6a), suggesting a possible role of MAO-A in promoting human macrophage immunosuppressive polarization. Time-course analysis of MDM culture confirmed an upregulation of MAO-A gene and protein expression during macrophage differentiation that was further upregulated post IL-4/IL-13-induced immunosuppressive polarization (Fig. 6b-d). Blockade of MAO-A using phenelzine significantly inhibited IL-4/IL-13-induced immunosuppressive polarization of MDMs, evidenced by their decreased expression of immunosuppressive markers (i.e., CD206 and CD273; Fig. 6e and Supplementary Fig. 6a) and signature genes (i.e., ALOX15 and CD200R1; Fig. 6f,g). Collectively, these in vitro data suggest that MAO-A is highly expressed in human macrophagesespecially during their immunosuppressive polarization, and that MAO-A blockade has the potential to reprogram human macrophage polarization.
To directly evaluate whether MAO-A blockade could reprogram human TAM polarization in vivo, we established a human tumour/TAM xenograft NSG mouse model. A375 human melanoma cells were mixed with monocytes sorted from healthy donor peripheral blood mononuclear cells (PBMCs), and s.c. injected into NSG mice to form solid tumours, with or without phenelzine treatment after inoculation (Fig. 6h). Phenelzine treatment effectively suppressed immunosuppressive polarization of human TAMs (gated as hCD45+hCD11b+hCD14+; Supplementary Fig. 6b), supported by their decreased expression of immunosuppressive markers (i.e., CD206 and CD273; Fig. 6i,j).
Next, we studied whether MAO-A blockade-induced human TAM reprogramming could impact human T cell antitumour reactivity, using a 3D human tumour/TAM/T cell organoid culture (Fig. 6k). NY-ESO-1, a well-recognized tumour antigen commonly expressed in a large variety of human cancers 77, was chosen as the model tumour antigen. An A375 human melanoma cell line was engineered to co-express NY-ESO-1 as well as its matching MHC molecule, HLA-A2, to serve as the human tumour target (denoted as A375-A2-ESO; Supplementary Fig. 6c,d). NY-ESO- 1-specific human CD8+ T cells were generated by transducing healthy donor peripheral blood CD8+ T cells with a Retro/ESO-TCR retroviral vector encoding an NY-ESO-1 specific TCR (clone 3A1; denoted as ESO-TCR); the resulting T cells, denoted as ESO-T cells, expressed ESO-TCRs and specifically targeted A375-A2-ESO tumour cells, thereby modeling the tumour-specific human CD8+ T cells (Supplementary Fig. 6e,f). Human MDMs were cultured from healthy donor PBMCs, followed by IL-4/IL-13 stimulation to induce immunosuppressive polarization in the presence or absence of phenelzine treatment (Fig. 6k). The A375-A2-ESO human melanoma cells,ESO-T cells, and IL-4/IL-13-polarized MDMs were mixed at a 2:2:1 ratio and placed in a 3D tumour organoid culture mimicking TME (Fig. 6k). IL-4/IL-13-polarized MDMs effectively suppressed ESO-T cell-mediated killing of A375-A2-ESO tumour cells; this immunosuppressive effect was largely alleviated by phenelzine treatment during MDM polarization (Fig. 6l). Accordingly, ESO-T cells co-cultured with phenelzine-treated MDMs, compared to those co- cultured with non-phenelzine-treated MDMs, showed an enhancement in T cell activation (i.e., increased cell number, increased CD25 expression, and decreased CD62L expression; Fig. 6m and Supplementary Fig. 6g). Collectively, these data suggest that MAO-A blockade-induced human TAM reprogramming has the potential to improve antitumour T cell responses.
To study MAOA gene expression in primary human TAMs, we collected fresh ovarian cancer tumour samples from patients, isolated TAMs (sorted as DAPI-hCD45+hCD11b+hTCRαβ-hCD14+ cells; Supplementary Fig. 6h), and assessed their MAOA gene expression. Primary human monocytes isolated from health donor PBMCs (sorted as DAPI-hCD45+hCD11b+hTCRαβ-hCD14+ cells; Supplementary Fig. 6i) were included as controls. Like mouse TAMs, human TAMs expressed high levels of MAOA gene, confirming MAO-A as a valid drug target in human TAMs (Fig. 1a and Fig. 6n).
Lastly, we conducted clinical data correlation studies to investigate whether intratumoural MAOA gene expression is correlated with clinical outcomes in cancer patients, using the TIDE computational method Intratumoural MAOA expression level was negatively correlated with patient survival in multiple cancer patient cohorts spanning ovarian cancer (Fig. 6o) 78, lymphoma (Fig. 6p) 79, and breast cancer (Fig. 6q) 80. Moreover, analysis of a melanoma patient cohort receiving anti-PD-1 treatment showed that high levels of intratumoural MAOA expression largely abrogated the survival benefit offered by the PD-1 treatment, suggesting that combining MAO-A blockade therapy with PD-1/PD-L1 blockade therapy may provide synergistic therapeutic benefits through modulating TAM polarization and thereby changing the immunosuppressive TME and improving antitumour immunity (Fig. 6r) 81. Of note, these whole tumour lysate transcriptome data analyses could not localize the MAOA expression to a specific cell type (e.g., TAMs); future studies of quality transcriptome data generated from single cells or sorted TAMs are needed to obtain such information. Nonetheless, the present clinical data correlation studies identified MAO-A as a possible negative regulator of survival in a broad range of cancer patients, including those receiving existing ICB therapies, suggesting MAO-A blockade as a promising avenue for developing new forms cancer therapy and combination therapy.
Taken together, these human TAM and clinical correlation studies confirmed MAO-A as a promising drug target in human TAMs and support the translational potential of MAO-A blockade for cancer immunotherapy through targeting TAM reprogramming.