GOT2: An Unexpected Mediator of Immunosuppression in Pancreatic Cancer

The glutamic-oxaloacetic transaminases (GOT), also called aspartate transaminases, are important enzymes that exist in two isoforms: GOT1 in the cytosol and GOT2 in the mitochondria. GOTs are primarily known to catalyze transamination, which is the transfer of amino groups from an α-amino acid (e.g., aspartate) to an α-keto carboxylic acid (e.g., α-ketoglutarate). This metabolic function enables the production and breakdown of amino acids, notably aspartate. GOTs also maintain cellular redox balance by facilitating the malate–aspartate shuttle, which transfers reducing equivalents between the cytosol and the mitochondria, enabling cells to overcome oxidative stress. Previous studies have shown that pancreatic cancer cells sustain rapid proliferation by hijacking both the transamination and redox balance functions of GOTs (1, 2). Besides these metabolic functions, little is known about GOTs. GOT2 has been associated with fatty acid binding and uptake, and, in clinical diagnostics, measurement of blood GOT level is part of biochemical tests for liver function.

In this Cancer Discovery issue, Abrego and colleagues (3) combined cutting-edge functional and pharmacologic studies to unravel a novel nuclear role for GOT2 in pancreatic cancer that is distinct from its metabolic function. The authors started by using the innovative CRISPR/Cas9 technique to delete GOT2 in multiple human and mouse pancreatic cancer cell lines, the latter derived from the commonly used KPC model (4), based on the pancreas-specific expression of mutant Kras and Trp53. In vitro, deleting GOT2 did not alter cancer cell proliferation. Upon transplantation in immunodeficient mice, GOT2-proficient and GOT2-knockout cells grew similarly. Surprisingly, when KPC GOT2-knockout cells were transplanted in syngeneic mice—thus, in the context of an intact immune system—they were unable to grow efficiently. This finding pointed to a non–cell-autonomous role of GOT2, possibly immune-mediated.

The authors thus analyzed the immune microenvironment in tumors expressing or lacking GOT2. They observed that GOT2-deficient tumors had reduced macrophage infiltration; they specifically lacked Arginase 1⁺ macrophages with likely immunosuppressive function. Conversely, tumors lacking GOT2 had increased lymphocyte infiltration, including CD8⁺ and CD4⁺ T cells. The increase in T-cell infiltration occurred early during tumor formation. Importantly, treatment with both CD4⁺ and CD8⁺ T cell–neutralizing antibodies rescued the growth of GOT2-deleted tumors. Thus, epithelial GOT2 expression is required to restrain antitumor immunity in a mouse model of pancreatic cancer. Supporting the mouse data, analysis of The Cancer Genome Atlas database revealed a positive correlation between GOT2 expression in human pancreatic cancer and gene expression programs linked to lymphocyte activation.

Next, Abrego and colleagues applied a combination of computational and molecular biology analyses to define the mechanism by which GOT2 expression in the tumor cells regulated immune responses. They discovered that GOT2 was localized to the nucleus of pancreatic cancer cells, where it enhanced fatty acid trafficking and binding to the PPARδ nuclear receptor. Accordingly, pancreatic ductal adenocarcinoma (PDAC) cells with GOT2 deletion had reduced PPARδ activity compared with PDAC cells expressing GOT2. Analysis of the crystal structure of human GOT2 and known fatty acid ligands for PPARδ confirmed that the GOT2–PPARδ interaction was mediated by fatty acid binding. Of note, the authors identified arachidonic acid as a prevalent GOT2 binding fatty acid. Arachidonic acid is a polyunsaturated fatty acid found on the plasma membrane of cells and is important for several biological functions, including cellular repair, inflammatory response, and neuronal development. Thus, a recurring theme is that PDAC cells take advantage of normal cellular processes to sustain their growth. By activating PPARδ, GOT2 regulated transcription of prostaglandin-endoperoxide synthase 2 (PTGS2), which encodes COX2, a known driver of immunosuppression (5), and is a mediator of pancreatic cancer progression in mice (6).

Concurrent to the study by Abrego and colleagues, two recent publications on GOT2 focused on its better known metabolic role. Garcia-Bermudez and colleagues (7) and Kerk and colleagues (8) inactivated GOT2 in a series of human pancreatic cancer cell lines. The two groups observed inhibition of cell growth in vitro but, like Abrego and colleagues, found no difference in growth in immunodeficient mice. Kerk and colleagues focused on mechanisms that allowed pancreatic cancer cells to bypass loss of GOT2 in vivo. GOT2 is necessary for aspartate biosynthesis from glutamine; in turn, aspartate is required for cell growth. For cancer cells to grow while lacking GOT2, alternative metabolic pathways must be at play. In vivo, cancer cells are surrounded by other components of the tumor microenvironment, such as fibroblasts; Kerk and colleagues observed that coculture with fibroblasts or with fibroblast-conditioned medium rescued growth of GOT2-null pancreatic cancer cells in vitro. Metabolic analysis revealed that fibroblasts provide pyruvate, which in turn is used by cancer cells for alternative biosynthesis of aspartate. To complement the transplantation studies, Kerk and colleagues generated KC mice lacking epithelial GOT2 expression. Intriguingly, KC;GOT2^f/f mice have similar progression rates to KC mice expressing GOT2, in apparent contrast with the Abrego study. Likely, differences in experimental design account for these differences: KC;GOT2^f/f mice lack GOT2 from the onset of carcinogenesis. Thus, both metabolic and immune adaptations are possible to bypass GOT2 dependency; identifying them will require further characterization of the models. Lastly, spontaneous tumors have a more abundant fibroblast population than transplanted tumors; whether fibroblasts mediate resistance to GOT2 ablation even in the context of an intact immune system remains to be addressed. Garcia-Bermudez and colleagues were particularly interested in the effects of GOT2 inactivation in pancreatic cancer cells grown in conditions of hypoxia—a key characteristic of the hypovascular microenvironment in pancreatic cancer. Garcia-Bermudez and colleagues observed that the growth defect caused by GOT2 inactivation was more pronounced in conditions of hypoxia; similar to Kerk, they observed that conditioned medium from pancreatic stellate cells, a type of pancreatic fibroblast, rescued the growth of GOT2-null pancreatic cancer cells, even in conditions of hypoxia. Garcia-Bermudez and colleagues also described an alternative mechanism to bypass GOT2 loss. In conditions of hypoxia, PDAC cells ramp up macropinocytosis through activation of hypoxia-inducible factor 1 subunit alpha (HIF1A). Macropinocytosis facilitates the scavenging of aspartate from albumin and ultimately circumvents the need for the aspartate that comes through GOT2.

An altered cellular metabolism and profound immunosuppression are defining features of pancreatic cancer. In this elegant study, Abrego and colleagues link the two by identifying a metabolic enzyme that also functions as a fatty acid transporter and mediator of an immunosuppressive gene expression program in pancreatic cancer; interestingly, their study is complemented by two recent articles assessing the metabolic role of GOT2 (Fig. 1). Abrego and colleagues describe a novel, non–cell-autonomous role of GOT2 in mediating the activation of COX2 and consequently induce the accumulation of immunosuppressive macrophages while inhibiting T-cell infiltration. In their model, GOT2 ablation is sufficient to restore antitumor immunity in a mouse model of pancreatic cancer. Important questions should be addressed prior to applying these findings to human patients. Unlike mice that are healthy until tumor cells are implanted, patients with pancreatic cancer have profound local and systemic immune suppression as well as T-cell populations that are largely dysfunctional (9); whether targeting GOT2 in preexisting tumor is sufficient to reverse immunosuppression remains to be addressed. Combination therapy approaches targeting myeloid cells together with immune-checkpoint blockade have also recently shown no benefit in the clinic (10), highlighting the need for new approaches. The current study suggests that GOT2 inhibition should be considered a therapeutic target in pancreatic cancer. Future preclinical work will be needed to test possible combination approaches. A second important consideration is that, given the heterogeneity of human tumors, stratifying patients to define the populations most likely to benefit from GOT2 targeting is imperative. Lastly, most targeted therapies elicit compensatory mechanisms, and metabolic drugs are no different. Identifying mechanisms of resistance and modifying treatment in response will be essential. Emerging pipelines, such as the Precision Promise clinical trial sponsored by the Pancreatic Cancer Action Network, propose a flexible trial design. Similar approaches, with bench to bedside followed by bedside to bench and reevaluation of therapy, are likely to become more common in the treatment of recalcitrant cancers.

No disclosures were reported.