A metabolic axis of immune intractability

Abstract

Immune cells in the tumor niche robustly influence disease progression. Remarkably, in cancer, developmental pathways are re-enacted. Many parallels between immune regulation of embryonic development and immune regulation of tumor progression can be drawn, with evidence clearly supporting an immune-suppressive microenvironment in both situations. In these ecosystems, metabolic and bioenergetic circuits guide and regulate immune cell differentiation, plasticity, and functional properties of suppressive and inflammatory immune subsets. As such, there is an emerging pattern of intersection across the dynamic process of ontogeny and the ever-evolving tumor neighborhood. In this article, we focus on the convergence of immune programming during ontogeny and in the tumor microenvironment. Exemplifying dysregulation of Hedgehog (Hh) activity, a key player during ontogeny, we highlight a critical convergence of these fields and the metabolic axis of the nutrient sensing hexosamine biosynthetic pathway (HBP) that integrates glucose, glutamine, amino acids, acetyl CoA, and uridine-5’-triphosphate (UTP), culminating in the synthesis of UDP-GlcNAc, a metabolite that functions as a metabolic and bioenergetic sensor. We discuss an emerging pattern of immune regulation, orchestrated by O-GlcNAcylation of key transcriptional regulators, spurring suppressive activity of dysfunctional immune cells in the tumor microenvironment.

Introduction

For tumors to develop successfully, they must effectively overcome the immunoediting process: (i)the elimination phase, where tumor cells are destroyed by immune cells; (ii) the equilibrium phase, where less immunogenic tumor cells are established and begin expanding; and (iii) the escape phase, where tumor cells successfully overcome the immune response and establish a tumor. Immunity is comprised of two arms, both of which are critical in tumorigenesis and tumor progression. Rapid nonspecific responses against pathogens are carried out by the innate arm of immunity, and slow, antigen-specific responses are accomplished through adaptive immunity. Innate immune responses are carried out mainly by cells derived from common myeloid progenitor cells, including dendritic cells (DCs), macrophages, and granulocytes, whereas cells derived from common lymphoid progenitor cells, namely T and B cells, predominately execute adaptive immune responses. The finely orchestrated functions of both arms are critical for limiting tumor growth and progression. There is an emerging pattern of intersection between the tumor microenvironment and the dynamic process of ontogeny. In this perspective, we will highlight the convergence of immunometabolic programming during embryonic development and in the tumor microenvironment.

The hexosamine biosynthetic pathway is a critical metabolic pathway that integrates carbohydrate and fatty acid metabolism in immune cells

Strategic orchestration of multiple metabolic pathways regulates immune cell differentiation, plasticity, and functional properties. The major metabolic and bioenergetic mechanisms include oxidative phosphorylation (OXPHOS), glycolysis, flux through the tricarboxylic acid cycle, pentose phosphate pathway, amino acid metabolism, fatty acid oxidation (FAO), and fatty acid synthesis. These metabolic circuits generate intermediates including fructose-6-phosphate, acetyl Coenzyme A (Acetyl CoA), glutamine, and uridine-5’-triphosphate (UTP) that are utilized in the hexosamine biosynthetic pathway (HBP). As a branch of glycolysis, the HBP produces the nucleotide sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is the substrate for O-GlcNAcylation, a post-translational modification of intracellular proteins that regulates protein stability and activity, and fine-tunes signal transmission of numerous pathways during embryonic development and in various immune cells [1]. The O-GlcNAc transferase (OGT) enzyme adds an O-GlcNAc unit to the serine or threonine residues of proteins, and O-GlcNAcase (OGA) reverses these processes [2]. The optimal functioning of this pathway is paramount in pregnancy, including during pre-implantation and implantation, and embryo development, and the dysregulation of HBP and O-GlcNAcylation leads to placental defects and embryotoxic effects [3]. In mouse embryos, OGA deficiency leads to aberrant chromosome segregation and aneuploidy due to incomplete cytokinesis, delayed embryo growth, and consequent death due to various abnormalities [4]. In the context of neurodevelopment, the activity of the cycling enzymes OGT and OGA is essential because their deficiency manifests as altered synaptic morphology, cognitive dysfunction, intellectual disability, and motor and sensory nerve dysfunction [5]. OGT deficiency has been demonstrated to inhibit neuronal development by mitigating WNT/β-catenin activity [6]. Notably, an increased flux through the HBP and O-GlcNAcylation characterizes several types of cancers and contributes to increased tumor cell proliferation, angiogenesis, and metastasis [7].

O-GlcNAcylation influences immune cell development and activity. In macrophages, O-GlcNAcylation has both positive and negative regulatory effects on their inflammatory response [8]. In experimental stroke models, hyper O-GlcNAcylation inhibits NF-κB p65 signaling, providing neuroprotection [9]. Myeloid-specific deletion of OGT increases NF-κB signaling and cytokine production in Toll-like receptor (TLR)-stimulated macrophages, and inhibition of O-GlcNAcylation suppresses lipopolysaccharide (LPS)-induced macrophage inflammatory responses [8]. O-GlcNAcylation also influences other myeloid subsets, including neutrophils, monocytes, and DCs. Increased O-GlcNAcylation in neutrophils promotes their migratory properties and chemotaxis [1]. This may be due to effects on multiple pathways, including PI3K, Rac, Rho family GTPase, p38 kinase, and ERK1/ERK2, which may be key players in O-GlcNAcylation-dependent regulation of neutrophil activity [1]. In human monocyte-derived dendritic cells (moDCs), OGT inhibition hinders the development of monocytes into immature DCs, decreasing their endocytic ability and IL10 expression, and increasing IL6 production [10]. Few studies have evaluated the function of O-GlcNAcylation in B cells, with a consensus that O-GlcNAcylation enhances B-cell activation and facilitates memory and antibody production [11].

In the context of O-GlcNAcylation, T cells are the most studied adaptive immune cells. OGA knockout decreases hematopoietic progenitor cells that differentiate into T- and B-cell populations, implying that dysregulated O-GlcNAcylation impairs overall immune cell development [12]. During the critical stages of T-cell development and differentiation, glutamine and glucose are used as an energy source to regulate intracellular protein O-GlcNAcylation. Furthermore, OGT acts as a checkpoint for Notch-mediated control of T-cell progenitors in the thymus, as well as a checkpoint for T-cell receptor (TCR)-mediated positive selection and peripheral T-cell clonal expansion [13]. A previous study demonstrates that O-GlcNAcylation of nuclear factor of activated T cells (NFAT) and p65 transcription factors modulate their transcriptional activity and contribute to T-cell activation and IL2 production [14]. In mice, TCR activity results in O-GlcNAcylation of the NF-kB subunit, c-Rel, at S350 in the nucleus, and diminishing this modification disrupts c-Rel-mediated expression of IL2, IFNγ, and colony-stimulating factor 2 (CSF2) in response to TCR activation [15]. These collective findings support the role of O-GlcNAcylation in modulating innate and adaptive immune responses in to the context of immune activation, antigen presentation, and cytokine expression.

Parallels in the immune system during embryonic development and tumor progression

Tumors usurp and reactivate developmental signaling pathways, including Cripto-1-Nodal, WNT/β-catenin, Notch, Hippo, TGFβ, and Hedgehog (Hh). Each of these pathways is evolutionarily conserved and, in a finely orchestrated manner, regulate cell fate determination, stem cell renewal, pluripotency, cell polarity, and motility. Aberrant activation of these pathways in tumor cells enhances proliferation, survival, epithelial to mesenchymal plasticity, and metastasis and is often correlated with worse patient outcomes. As such, there is an emerging pattern of intersection across the dynamic process of ontogeny and the ever-evolving tumor microenvironment.

The first sign of immunity during embryogenesis occurs in the yolk sac mesoderm and extraembryonic mesenchymal tissue as early as 3–4 weeks. This is characterized primarily by the generation of hematopoietic stem cell progenitors, natural killer (NK) cell progenitors, mast cells, primitive macrophages, and innate lymphoid cell (ILC) progenitors [16]. During early stages of development, tolerogenic and protective immune responses are critical to ensure the tight regulation of developing inflammatory cells. Innate tissue inducer cells are necessary to promote tissue protection and remodeling [17]. During the second month of gestation, regulatory T cells (Tregs) play an important role in hindering the activity of inflammatory cells and are present in high abundance in peripheral lymphoid organs [18]. This can be explained by elevated levels of fetal TGFβ and naïve CD4⁺ T cells with a transcriptome and epigenome that overlaps with that of Tregs and, therefore, are poised to differentiate into the Treg lineage [19]. Similarly, naïve CD4⁺ T cells found in fetal cord blood are marked by high expression of the Th2 lineage commitment factor GATA3 and the Th2 cytokines IL4 and IL13 [20]. CD8⁺ T cells within cord blood exhibit a much lower cytotoxic response compared to adult CD8⁺ T cells, but are capable of responding to viral infections and form a memory population [18]. Although innate immune cells, including macrophages, NK cells, neutrophils, and DCs have been reported in the fetus, they are characterized by poor function and diminished inflammatory responses [21]. As such, sustained suppressive immunity is critical during embryonic development.

Evidence clearly supports a common immune-suppressive microenvironment between the developing embryo and the tumor ecosystem. Tumor cells and embryonic cells are both characterized by proliferative capabilities, stemness attributes, and the ability to co-opt immune escape mechanisms to support a common tolerogenic environment [22]. One mechanism includes an upregulation of TGFβ found in several epithelial cancers to induce Treg and suppressive neutrophil expansion within the TME [23]. Moreover, Human Leukocyte Antigen-G (HLA-G) is upregulated in the developing embryo and secreted from the tumor within extracellular vesicles. HLA-G suppresses adaptive and innate components of the immune system, including cytotoxic T cells, NK cells, DCs, and B cells [24]. Many parallels between immune regulation of embryonic development and immune regulation of tumor progression can be drawn, with evidence clearly supporting an immunosuppressive microenvironment in both situations. Exemplifying dysregulation of Hh activity, a key player during ontogeny, we highlight a critical convergence of these fields and the metabolic axis of the nutrient sensing HBP.

Hedgehog signaling governs an immune-suppressive metabolic program

Hh signaling (Figure 1) [25] is activated when one of three Hh ligands, Sonic Hedgehog (SHH), Indian Hedgehog (IHH), or Desert Hedgehog (DHH), binds to the canonical receptor, Patched (PTCH1), a 12-span transmembrane protein resulting in de-repression of the signaling transducer, smoothened (SMO). SMO, a GPCR-like protein, accumulates in the primary cilium and releases the transcription factor, glioma-associated oncogene homolog (GLI), from suppression by kinesin family protein 7 (KIF-7) and suppressor of fused (Sufu), allowing GLI in its active form to translocate to the nucleus and initiate transcription of target genes.

Figure 1. The Hedgehog signaling pathway.

The Hedgehog (Hh) signaling pathway is activated when Hh ligands bind to the canonical transmembrane receptor, Patched (PTCH1), a 12-span transmembrane protein. In the absence of Hh ligand, PTCH continues to suppress smoothened (SMO), and Sufu continues to suppress glioma-associated oncogene homolog (GLI) and restrain it in the cytoplasm. Sufu recruits protein kinase A (PKA), Beta-transducin repeats-containing proteins (β-TRcP), Glycogen synthase kinase-3 beta (GSK3-β), and casein kinase 1 (CK1), which phosphorylate and ubiquitinate GLI, making it repressive (Gli-R). SMO is downregulated when Hh ligand (Hh-L) binds to PTCH, Growth Arrest Specific 1 (GAS1) and Cell Adhesion Associated, Oncogene Regulated (CDON). SMO accumulates in the primary cilium and releases the GLI transcription factor from KIF-7 and Sufu, allowing GLI to translocate to the nucleus and activate (Gli-A) transcription of target genes. Inhibition of the Hh pathway with a SMO inhibitor (SMOi) prevents GLI from translocating to the nucleus.

Hh signaling was first demonstrated to be critical during thymocyte differentiation from CD4⁻CD8⁻ double-negative (DN) thymocytes to CD4⁺CD8⁺ double-positive (DP) thymocytes [26]. This finding underscored the potential role of Hh signaling in immunoregulation. Subsequent studies have probed the role of Hh signaling in adaptive and innate immunity. The role of Hh signaling in B-lymphocyte biology has not been extensively studied, but there are data indicating that GLI3 promotes B-cell development and protects germinal center B cells from apoptosis [27]. In the context of adaptive immunity, an immunosuppressive pattern of Hh regulation is emerging. Hh/GLI activity induces IL4 expression, promoting Th2 differentiation and allergy responses [28], and in Tregs, enhances expression of TGF-β and forkhead box P3 (FOXP3) [29]. In a genetically modified mouse model in which PTCH is inactivated (leading to overactivation of Hh signaling), mice demonstrate a better response to experimental autoimmune encephalomyelitis (EAE) through a systemic increase in the production of immunosuppressive cytokines, IL10 and IL4, by CD4⁺ T cells [30]. Interestingly, in CD8⁺ T cells, Hh signaling promotes Rac1 synthesis, priming CD8⁺ cytotoxic T cells for granule release [31], and in innate-like lymphocytes, Hh signaling promotes differentiation of gamma-delta (γδ) T cells [32] and natural killer T cells [33]. In innate immune cells, Hh signaling enhances signal transducer and activator of transcription 6 (STAT6) activity by promoting immunosuppressive macrophage polarization [34], and induces the expression of programmed death ligand 1 (PD-L1) in antigen-presenting DCs and tumor-associated macrophages [35]. In the stomach, Hh/GLI supports immune-suppressive granulocytic myeloid suppressor cell differentiation [36].

The OXPHOS bioenergetic program plays a key role in contributing to enhanced suppressive activity of macrophages and Tregs by meeting their high energy demands. Hh blockade reduces OXPHOS and FAO in immune-suppressive macrophages and Tregs. Moreover, in suppressive macrophages, Hh inhibition upregulates glycolysis, prompting a bioenergetic state that is reminiscent of inflammatory, tumor-reactive macrophages. A role for Hh signaling in influencing a common metabolic cascade in Tregs and immune-suppressive macrophages is beginning to emerge (Figure 2). In particular, both cell types harness the O-GlcNAc pathway to bolster the activity of pivotal transcription factors that define their immunosuppressive functions. The identification of a GLI binding site in the Ogt promoter sequence indicates that Hh activity directly enhances cellular O-GlcNAcylation [37]. Whereas O-GlcNAcylation of STAT6 solidifies the immune-suppressive M2 macrophages with enhanced transcription of Mrc1 and Arg1 and promotes the transcriptional activity of FOXP3 in Tregs [37, 38], Hh blockade diminishes O-GlcNAc post-translational modifications on these key transcription factors, alleviating their immune-suppressive functions. In Tregs, Hh inhibition also decreases O-GlcNAc modification of signal transducer and activator of transcription 3 (STAT3), a master transcription factor for the Th17 lineage. In contrast to STAT6 and FOXP3, wherein decreased O-GlcNAcylation mitigates their transcriptional activity, decreased O-GlcNAcylation of STAT3 leads to enhanced transcription activity and expression of key Th17 factors such as Rorc and Il17a in Tregs, thus promoting their trans-differentiation into Th17 cells [38]. As such, by exerting a direct transcriptional influence on Ogt, Hh executes a multimodal metabolic control on the immune-suppressive activity of macrophages and Tregs. Collectively, Hh blockade initiates convergent metabolic and bioenergetic re-wiring in immune-suppressive macrophages and Tregs resulting in diminished suppressive functionality and an enhanced pro-inflammatory niche.

Figure 2. Hedgehog signaling metabolically rewires M2 macrophages and Tregs to enhance their suppressive phenotypes and functions.

Tumor cells secrete Hh ligand (Hh-L), which activates Hedgehog (Hh) signaling and promotes O-GlcNAcylation of FOXP3 and STAT6 in Tregs and M2 macrophages, solidifying their suppressive activity. Furthermore, Hh signaling promotes two key bioenergetic programs, oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO), that aid M2 macrophages and Tregs in their suppressive functions. Inhibiting Hh reduces O-GlcNAcylation of key transcription factors, as well as OXPHOS and FAO, attenuating the suppressive activity of Tregs and M2 macrophages. Hh blockade induces metabolic changes that promote upregulation of inflammatory functions and the transformation of M2 macrophages to M1-like phagocytic macrophages and Tregs into inflammatory Th17-like T cells, all of which contribute to the establishment of a tumor-eradicating microenvironment. GZMB: granzyme B.

Conclusion and future considerations

A critical role for Hh signaling in ontogeny and embryonic development has been well-established over the past several decades and aided in the characterization of aberrant Hh signaling in promoting tumorigenesis, invasion, and metastasis through a direct impact on tumor cells. However, there is a clear parallel in the immune environment during embryonic development and the tumor immune microenvironment, both of which are dominated by suppressive immune cell types. Appreciating the pivotal role of cellular cross-talk in both ontogeny and tumor progression, it is critical to study the impact of dysregulated developmental signaling pathways, such as the Hh pathway, on neighboring immune and stromal cells to gain a comprehensive understanding that will inform cancer therapeutic options. Consequently, lessons from developmental biology are critical not only to understand tumor biology, but also for contextualizing the complex tumor microenvironment.

Most cancer treatment studies have focused predominantly on the effects of therapies on tumor cells, with little appreciation for stromal and immune cells in the complex tumor ecosystem. This has historically been the case with the FDA-approved Hh inhibitor Vismodegib, which is used for the treatment of basal cell carcinoma [39]. However, few studies have assessed the role of Hh signaling in immunity, and even fewer have assessed the role of Hh signaling in immune cells in the complex TME.

Metabolic and bioenergetic circuits critically guide and regulate immune cell differentiation, plasticity, and functional properties. In fact, the TME has a unique metabolic landscape that executes an immunosuppressive program. As a mechanism that acutely integrates and senses the nutrient status of the cells as a metabolic rheostat, the HBP modulates cellular energetics and metabolism and governs important transcriptional programs in immune cells, presenting an important metabolic axis that can program immune cell flexibility. Although HBP modulation presents as an attractive therapeutic target to restore immune functions, employing this strategy will involve several considerations ranging from neurological, musculoskeletal, hematopoietic, and other systemic effects, since the pathway is critical for homeostasis in several tissues and organs. On the other hand, pharmacological inhibition of Hh reprograms an immunologically cold tumor milieu to be immune-reactive. Unlike FDA-approved immune checkpoint blockade therapies that target T cells, Vismodegib re-sculpts the innate and adaptive arms of the immune system by impinging on the HBP metabolic axis. Being able to pivot this axis of immune intractability to generate a tumor eradicating microenvironment will also set the foundation for improving the efficacy of immunotherapy for cancer patients or inform therapeutic options for patients who develop secondary resistance to these therapies.