The missing link? LGMN connects hypoxia and immunosuppression in glioblastoma

Abstract

In this issue, Pang and colleagues¹ identify the protease legumain as a potential immunotherapy target in glioblastoma that drives tumor-associated macrophages in response to hypoxia.

In this issue, Pang and colleagues¹ identify the protease legumain as a potential immunotherapy target in glioblastoma that drives tumor-associated macrophages in response to hypoxia.

Main text

Immunotherapies have limited efficacy in glioblastoma (GBM), the most common primary malignant brain tumor, in part due to the immune suppressive environment in the brain. Moreover, a hallmark of the GBM tumor microenvironment (TME) is hypoxia, which also contributes to cancer immune evasion.² This is partially a result of enhanced immunosuppressive tumor-associated macrophages (TAMs), which constitute a large portion of GBM-infiltrating cells.³ While both hypoxia and TAM-mediated immunosuppression are associated with poor therapeutic responses, there is a limited understanding of the molecular pathways that facilitate the communication between these two mechanisms.

Despite the prevalence of TAMs in GBM, depletion strategies have shown modest clinical efficacy.⁴ It is becoming increasingly evident that signaling between cancer cells and TAMs produces context-dependent TAM states, which promote tumor progression and therapeutic resistance.⁵ These signals include cytokines, growth factors, and matrix remodeling proteins. Secreted proteases have also been associated with cancer progression across multiple cancer types, and inhibition of proteases has been demonstrated to suppress GBM tumor growth in mice.⁶ How specific proteases interact with the TME and immune cells is less clear. Here, the authors explore the role of extracellular proteases in the context of the hypoxic and immunosuppressive GBM microenvironment and identify legumain (LGMN) as a treatment target (Figure 1).

Figure 1.

Blockade of the HIF1α-LGMN axis inhibits tumor growth and synergizes with anti-PD1 in GBM

Initially, the authors established that LGMN is enhanced in GBM-associated macrophages by using RNA sequencing (RNA-seq) data of GBM-associated bone marrow-derived macrophages (BMDMs) from mouse models and microarray data from patient TAMs. They observed that treatment of mouse macrophage-like cell line, a human macrophage cell line, and mouse BMDMs with recombinant LGMN increased the expression levels of immunosuppressive markers arginase 1 (ARG1), vascular endothelial growth factor A (VEGFA), and CD206. Conversely, LGMN inhibitor treatment and small hairpin RNA (shRNA)-mediated depletion of LGMN reduced the percentage of CD206+ and ARG1+ macrophages, further confirming the specificity of the observed effect. These findings were supported in vivo with an orthotopic GBM model by using an LGMN inhibitor, which demonstrated that LGMN promotes polarization of TAMs toward an immunosuppressive phenotype in GBM. The authors then investigated potential downstream pathways that mediate the LGMN-induced immunosuppressive macrophage polarization. Upon LGMN recombinant protein treatment of mouse and human macrophage-like cell lines and BMDMs, the p-GSK-3β and p-STAT3 pathways were upregulated. Hyperactive p-STAT3 promotes the expression of immunosuppressive factors, such as VEGF, and p-GSK-3β contributes to immunosuppression by promoting pathways that inhibit apoptosis.⁷ The use of both pharmaceutical inhibitors against these two pathways and genetic knockdown showed a significant impairment of LGMN-induced upregulation of ARG1 and CD206 in mouse and human macrophage-like cell lines, exemplifying that GSK-3β-STAT3 signaling mediates the LGMN-directed polarization in macrophages.

To further examine the factors that inform LGMN activity, the authors analyzed The Cancer Genome Atlas dataset and observed a positive correlation between LGMN with the hypoxia-associated gene signatures in GBM. Because hypoxia-inducible factor 1-alpha (HIF1α) is a master hypoxia-regulated transcription factor,⁸ the authors interrogated the relationship between HIF1α and LGMN in macrophages and found that both human and mouse LGMN promoters contained HIF1α binding sites. This finding suggested that LGMN can be a direct target of hypoxia signaling. In vivo, this relationship was exemplified because myeloid-specific HIF1α knockout (HIF1α-mKO) mice exhibited lower LGMN expression in BMDMs compared with the wild-type (WT) littermates. Similarly, pharmaceutical inhibition shRNA-mediated depletion of HIF1α reduced LGMN mRNA levels in a mouse macrophage-like cell line in vitro. These findings suggested that HIF1α directly regulates the expression of LGMN in macrophages. Furthermore, reduction in the number of CD206 or ARG1+ BMDMs caused by the genetic deletion of HIF1α was rescued by treatment with LGMN recombinant protein, suggesting that LGMN is downstream of HIF1α.

The authors next sought to test the therapeutic value of targeting LGMN signaling in preclinical GBM models. The frequency of LGMN and CD206 expressing TAMs was significantly reduced in GL261 and CT2A tumors in HIF1α-mKO mice compared with WT. Similar results were observed in LGMN-mKO mice, pointing to the potential role of the HIF1α-LGMN axis as a molecular target. Furthermore, pharmaceutical inhibition of LGMN and depletion of myeloid cells with anti-CSF-1R had a similar effect on the survival outcomes in CT2A-bearing mice, suggesting that LGMN effectively targets immunosuppressive myeloid cell populations. Immunosuppressive myeloid cells have been linked to resistance to PD-1. Therefore, the authors evaluated whether inhibition of HIF1α-LGMN signaling can prime anti-PD-1 response. Both pharmaceutical inhibition of LGMN and genetic KO of HIF1α further improved survival outcomes when combined with anti-PD-1.

The results of this study align with prior reports, which demonstrate that downregulation of LGMN in macrophages promotes tumor senescence in melanoma and breast cancer models.⁹ While the authors have previously proposed a therapeutic approach targeting microglia by blocking CLOCK-regulated LGMN in patient subsets with CLOCK amplification,^10,11 the current study offers a more broadly applicable target through the identification of a HIF1α-LGMN axis. Overall, the study highlights promise for a new therapeutic axis in GBM. However, additional work is needed to delineate the mechanism by which LGMN activates GSK-3β-STAT3 signaling in macrophages and to determine whether inhibition of this signaling axis is sufficient to reduce immunosuppressive TAM polarization and GBM growth. Finally, LGMN has been linked to other TME changes including tumor invasion, vascularization, and even p53 signaling. Future work may evaluate whether the HIF1α-LGMN pathway in TAMs can modulate additional microenvironmental changes in GBM.

In summary, understanding the complexity of the GBM TME is critical for developing effective treatment strategies. A combination of tumor hypoxia, myeloid cell-induced immunosuppression, and extracellular pro-tumorigenic signals have limited the successful implementation of immunotherapies in GBM. In this work, Pang and colleagues have identified LGMN and associated signaling pathways as key contributors to TAM-mediated immunosuppression. Improved anti-tumor effects of combined LGMN and PD-1 inhibitors offer promise for synergistic approaches that can improve the immunotherapy response in GBM.