Glutamine metabolism plays an important role in T-cell activation and proliferation, as a precursor for biosynthesis and source of ΑKG for the TCA cycle and ATP production (17, 30, 49). Glutamine induces a dose-dependent increase in proliferation of stimulated T-cells in vitro (23, 24). On the surface therefore, it may seem that glutamine inhibition would frustrate anticancer immunity, but recent investigation shows otherwise.

Johnson et al. demonstrated that in effector T-cell populations with transient GLS deficiency, IL2 promotes Th1 phenotype and CD8 T-cell function, suggesting that inhibition of glutamine metabolism enhances an anti-tumor immune composition (24, 50). Aberrant tumor metabolism, which also upregulates glutamine metabolism for biosynthesis and anaplerosis, depletes glutamine in the tumor environment (13, 24, 30). Inhibition of GLS with CB-839, or of glutamine metabolism broadly with JHU083 or DRP-104 (Sirpiglenastat, another pro-drug of DON), restores balance between tumor and T-cell glutamine utilization. Multiple authors have shown that CB-839, JHU083 and DRP-104 reduce tumor glutamine consumption and increase glutamine concentrations systemically and in the TME, providing T-cells with greater access to this conditionally essential amino acid (13, 17, 23, 24, 26, 29). CB-839 also has minimal impact on T-cell proliferation and acceptable toxicity profile, unlike the non-specific inhibitor DON (23, 24). Leone et al. found that JHU083 increased proliferation and activation markers in CD8+ T-cells in mice harboring MC38 colon cancer, and selective activation of this prodrug in the TME mitigates toxicity observed with DON (13).

Gross et al. demonstrated in a syngeneic CT26 colon cancer mouse model that combination CB-839 and anti-PD-1 or anti-PD-L1 therapy enhanced tumor regression and improved survival compared to control or monotherapy (23). These benefits were reversed with depletion of CD8+ T-cells from the tumors, supporting the hypothesis that GLS inhibition with CB-839 enhances CD8+ T-cell activity in the TME when administered with checkpoint inhibitors (23). These results led to the phase I/II trial CX-839-004; CB-839 with nivolumab in advanced melanoma, renal cell carcinoma (RCC), or non-small cell lung cancer. The trial completed recruitment in 2020. Preliminary results showed no toxicity above nivolumab alone, and three patients with melanoma and progression on prior immunotherapy achieved objective response (29).

Building on the potential to revitalize checkpoint inhibitor response in the pre-treated setting, Varghese et al. report promising preclinical results in treatment-naïve melanoma models (24). In mice vaccinated with melanoma-specific CD8+ T-cells and stimulatory molecules, CB-839 increased T-cell proliferation and activation compared to vehicle control (24). T-cell vaccination with CB-839 treatment led to the greatest decrease in B16 xenograft tumor growth vs. monotherapy or control. Mouse survival was 100% at 35 days in the combination group (24). These results suggest clinical study of GLS inhibition together with T-cell vaccines or other T-cell therapies is warranted.

Varghese et al. also tested CB-839 together with immune-checkpoint inhibitors in a BRAF V600E, high mutational burden melanoma model. Anti-PD-1 or anti-CTLA4 therapy inhibited tumor growth, in keeping with prior findings (24, 51). CB-839 monotherapy had no effect on tumor growth, but combination with anti-PD-1 or anti-CTLA4 showed synergistic effects, with triple therapy leading to apparent complete response at 29 days (24).

JHU083 has also shown exciting results, with improved tumor control and survival in mouse models of several tumor types (13). JHU083 with concurrent anti-PD-1 therapy in a MC38 mice showed complete response rate of ≥90%, compared to no complete responses with anti-PD-1 monotherapy (13). Lack of single-agent activity is a criticism some have leveled against CB-839 (13, 23, 52). In contrast, in one experiment, 2 of 5 mice with MC38 tumors had complete response to JHU083 monotherapy maintained >80 days after tumor injection (13). Additionally, 13 of 15 animals with complete response to JHU083 monotherapy rejected tumor upon rechallenge. These results indicate JHU083 may have single agent utility. As with the CB-839 experience above, depletion of T-cells demonstrated that JHU083 efficacy relied on CD8+ T-cell activity (13). Based on these data, along with RNA-seq and gene set enrichment analysis (GSEA) of tumor infiltrating lymphocytes (TILs), Leone et al. concluded that JHU083 pushes TILs toward a long-lived, memory-like, proliferative and highly activated effector phenotype (13, 17).

DRP-104 produced similar results in mice harboring colon and hepatocellular model tumors (26–28, 30). Yokoyama et al. found that DRP-104 tumor growth inhibition at day 12 in CT26-bearing mice was 48% with anti-PD-1, 90% with DRP-104 and 94% with combination (26, 27). In MC38-bearing mice, DRP-104 monotherapy led to growth inhibition of 96-101% and increase in median survival from 13 days with vehicle control to 31-38 days with DRP-104 (26). Eight of 16 CT26 mice treated with combination DRP-104 and anti-PD-L1 were tumor free at day 77, and all eight rejected tumor rechallenge (27). In mice with H22 hepatocellular tumors, DRP-104 at low (45 days) or high dose (47 days) alone and in combination with anti-PD-L1 blockade (76.5 or 94 days) significantly extended survival compared to vehicle (27.5 days) and anti-PD-L1 (29.5 days) alone (26, 27). There were 50% durable cures in high dose combination-treated mice (26, 27).

These data further support GLS and glutamine inhibition in combination with checkpoint blockade in the clinical, treatment-naïve setting. Additional study in combination with radiation also seems promising, since restoration of T-cell response could turn radioresistance into radiosensitivity.

While cancer cell metabolism hoards nutrients such as glutamine, the TME is further stripped of resources by the activity of M2 macrophages and MDSCs. Tumor-associated macrophages (TAMs) contribute to cancer progression and metastasis by promoting angiogenesis, invasion, motility, intravasation and extravasation, while also curating an immunosuppressive environment (25, 39, 53, 54). M2 TAMs and MDSCs express immune checkpoint molecules like PD-L1 to attenuate T-cell response, and metabolic enzymes which deplete nutrients and smother T-cell proliferation. Oh et al. report \ inhibition of glutamine metabolism with JHU083 promoted generation of antitumor, proinflammatory TAMs and reduced generation and recruitment of MDSCs (25). Similarly, Yokoyama et al. report that DRP-104 treatment increased M1-polarized TAMs and decreased MDSCs (26, 28).

4T1 is a triple-negative breast cancer model that is resistant to immune checkpoint blockade due to abundant suppressive TAMs and MDSCs (25, 55). JHU083 showed significant inhibition of 4T1 tumor growth in mice compared with vehicle, anti-PD1, anti-CTLA4 or combination checkpoint blockade (25). JHU083 also significantly inhibited lung metastasis. JHU083 enhanced checkpoint blockade activity in EO771 immunotherapy-sensitive tumors, and sensitized 4T1 immunotherapy-resistant tumors to checkpoint blockade (25).

Oh et al. demonstrated that modulation of TAMs and MDSCs is responsible for these effects. JHU083 decreased MDSCs at the primary tumor site and in the lungs (common metastatic site). This was accompanied by decreased MDSC recruitment and growth factors such as M-CSF, GM-CSF and G-CSF. Finally, Oh et al. found through RNA-seq and GSEA that JHU083 reprogrammed TAMs promoting an M1, antitumor phenotype via down regulation of immunosuppressive genes such as Il10 and Nos2, with upregulation of lysosome, Toll-like receptor and proinflammatory gene transcription.

These data suggest broad glutamine antagonism with JHU083 or DRP-104 may increase radiosensitivity by favoring an antitumor immune response, but clinical study including combination with radiation is necessary.

Sequencing of immunotherapy and radiotherapy is an area of ongoing research; full exploration of this topic is beyond the scope of this mini-review (56, 57). Optimal timing depends on the mechanism of immunotherapy agent used and the fraction size of radiation (57). Notably, secondary analysis of KEYNOTE-001 found a survival benefit with pembrolizumab in patients previously treated with radiotherapy vs no prior radiotherapy (56, 58). As such, several clinical studies have specified immunotherapy within 3-84 days after radiation (59–61). Radiation can begin before, during or after immunotherapy in the ongoing A082002 trial in metastatic NSCLC, as long as it begins within 60 days of registration (NCT04929041). We hypothesize that radiation followed by or concurrent with immunotherapy, rather than starting with immunotherapy, will lead to optimal results by exposing tumor antigens and utilizing radiation-induced increase in PD-L1 expression to fuel immunotherapy response (56, 62). This hypothesis seems to be supported by the survival benefit from pembrolizumab after radiation in the PEMBRO-RT trial, but only in patients with negative PD-L1 at baseline, where perhaps PD-L1 expression was induced by radiation (59).

Adding glutamine antagonists leaves additional uncertainty regarding the best sequence, but Leone et al. report that “concurrent, not sequential” administration of JHU083 and anti-PD-1 was most effective (13). Most pre-clinical and clinical studies have initiated glutamine antagonists concomitantly with immunotherapy (23–25, 29), others have used concomitant and sequential approaches without direct comparison of the two (24, 27, 28). Wicker et al. introduced CB-839 two days before radiating HNSCC cells (21).