Targeting Oxidative Phosphorylation to Increase the Efficacy of Radio- and Immune-Combination Therapy

Tumors quickly outgrow their vasculature, thereby limiting the amount of oxygen available for diffusion into the tissue in areas distant from vessels, while oxygen demand is also increased in proliferative tissues (1). The diffusion-limited deprivation of oxygen is known as chronic hypoxia, which occurs in most solid tumors. Moreover, the chaotic and unreliable nature of the tumor vasculature can also result in a more acute form of hypoxia by the temporal impaired blood flow through these vessels (Fig. 1). Hypoxia results in metabolic changes and hypoxic tumor cells are characterized by an increased glycolytic metabolism (2). Glycolysis leads to accumulation of glycolytic metabolites, such as lactate, and subsequent acidification of the tumor microenvironment (TME; ref. 2). Hypoxic areas in solid malignancies are highly therapy resistant (3, 4). A well-known example is hypoxia-mediated resistance to radiotherapy, primarily caused by the reversion of DNA-free radicals under hypoxia, whereas this DNA damage is fixed under normoxia (oxygen fixation hypothesis; ref. 5). Furthermore, radiation produces reactive oxygen species (ROS) that interact with DNA, inducing mitotic cell death. These ROS cannot be formed when oxygen is deprived, resulting in radioresistance (5). Several studies describe how radiotherapy can be enhanced by the alleviation of hypoxia, both in preclinical models, as well as in clinical studies (6–8). Another possible reason for the failure of radiotherapy is hypoxia-mediated immune escape, as a substantial part of the efficacy of radiotherapy relies on a durable anticancer immune response (9). This is, for instance, mediated by the depletion of immune effector cells from hypoxic areas and acidification of the TME (Fig. 1; refs. 10–12).

In this review, we will describe the mechanisms by which hypoxic stress causes immune suppression, involving different immune components, and how this contributes to treatment resistance. There are strong indications that by alleviating hypoxia and concomitant immune suppression, resistant tumor areas can become sensitive to treatment (13, 14). An effective way to accomplish this goal is the reduction of the oxygen consumption in tumors, hereby normalizing oxygen levels in remote tumor areas. Reduction of the oxygen consumption rate can be achieved by pharmacologic inhibition of oxidative phosphorylation (OXPHOS). As opposed to the longstanding notion that tumors are primarily glycolytic, including under aerobic conditions, as postulated by the Warburg effect, it has recently been described that OXPHOS remains active in many cancers and is a potential therapeutic target (15). Many important oncogenic pathways rely on mitochondrial metabolism and metabolic plasticity is important for tumor progression at every stage of development (16). For instance, in B16 melanoma, the Warburg effect has proven to be dispensable all together because of an upregulated mitochondrial metabolism (17). Current studies indicate that metabolic reprogramming might be an important strategy in reactivating the immune response and describe how this ultimately can also lead to an increased efficacy of the combination of immuno- and radiotherapy (18).

To counter hypoxic stress, several mechanisms are activated, such as the hypoxia-inducible factor 1 (HIF1) pathway (19), the unfolded protein response, and the mTOR pathway (20). The hypoxia response affects tumor metabolism, angiogenesis, pH regulation, and also impacts antitumor immunity (2). Immune effector cells can be influenced directly by hypoxia; for instance, expression of HIF1α (the O₂ regulated subunit of HIF1) leads to a transcriptional responses affecting immune cell metabolism and differentiation (21). Immune effector cells can also be affected indirectly via the release of immunosuppressive molecules (e.g., adenosine) by surrounding tumor cells and/or cancer-associated fibroblasts (22, 23).

Metabolic changes can be important drivers of the immune system, often governing the action and development of different immune cell subsets (24). Different populations of immune cells respond differently to hypoxic stress. Many effector cells that are important for tumor eradication, like CD8⁺ and CD4⁺ T cells, dendritic cells (DCs), and natural killer (NK) cells are suppressed (25) and less present in hypoxic areas (Figs. 1 and 2). The HIF1 transcriptional hypoxia response directly prevents activation of CD8⁺ T cells and CD8⁺ T cells are mostly absent from hypoxic areas in a syngeneic fibrosarcoma model (12, 26). Reduction of hypoxia can also restore the infiltration of CD3⁺ T cells in a spontaneous prostate cancer model in mice (27). On the other hand, immunosuppressive cell types, like tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs), are often overrepresented and overly active in hypoxic tumor areas (Fig. 2; ref. 25). In mice, hypoxia induces the accumulation of TAMs in 3LL lung and 4T1 mammary tumors (11). Hypoxia-associated activation of the HIF1 pathway in tumor cells indirectly promotes the accumulation of MDSCs in Hepa1-6 hepatocellular cancer via the extracellular conversion of ATP to 5′-AMP (28), while it directly promotes Treg activation in induced colon cancer (29). Acidosis of the TME, which is the consequence of hypoxia-induced glycolytic metabolism, has an immunosuppressive effect as well (10). Acidification of the TME in B16 melanoma skews macrophage polarization toward a noninflammatory phenotype, promoting tumor growth (30). In contrast, hypoxia can promote antitumor immunity in some cases. In GL261 glioblastomas, for example, although associated with an enhanced animal survival, HIF1α-deficient Tregs showed enhanced CD8⁺ T-cell–suppressing activity (31). Furthermore, hypoxic cell culture enhances the cytolytic activity of CD8⁺ T cells, while their capacity to produce cytokines is potentiated in hypoxic CT26 colorectal cancer (32–34). This highlights that the effect of hypoxia on the immune system can be heterogeneous within tumors and, being either direct or indirect, may likely be tumor type dependent.

In addition to the suppressing effects on immune effector cells, hypoxia can also increase the intrinsic resistance of tumor cells themselves to antitumor immune responses. This is, for instance, mediated by increased expression of cell-surface immune checkpoint molecules, like programmed death-ligand 1 (PD-L1) on tumor cells, which have emerged as promising therapeutic targets (35). Programmed death-protein 1 (PD-1) is expressed on activated T cells and functions as a feedback mechanism to protect against tissue pathology–induced autoimmunity upon binding to its ligand PD-L1 (36). Furthermore, HIF1α has been identified as a direct regulator of PD-L1, suggesting that combining hypoxia targeting with PD-L1 blockade may improve treatment outcome (37). Another therapeutically targeted immune checkpoint molecule, cytotoxic T-lymphocyte–associated protein 4 (CTLA-4), is under control of the adenosine-A2A adenosine receptor pathway in Tregs (38). As adenosine accumulates in hypoxic tumors (22), CTLA-4 upregulation has a potential role in hypoxia-mediated immune evasion as well. Expression of other immune checkpoint molecules, including VISTA, TIM-3, and LAG3, targeting of which is currently under clinical investigation, is regulated by hypoxia as well, contributing to immune repression in hypoxic areas (Fig. 2; refs. 34, 39, 40). As opposed to immune checkpoints regulating the adaptive immune response, the don't eat me signal CD47, which mediates resistance against innate immunity through the prevention of phagocytosis by macrophages, is also upregulated under hypoxic conditions as the CD47 gene is under direct control of HIF1α in breast cancer in vitro (41).

Tumor hypoxia and the presence of immunosuppressive cell types can be major obstacles for effective immunotherapy (42, 43). In a murine Braf^V600E-induced/Pten-deficient melanoma model, tumor hypoxia caused exhaustion of CD8⁺ T cells, acting as a barrier to PD-1 blockade immunotherapy (44). Also, the immunosuppressive properties of the metabolite adenosine, which is excessively produced in hypoxic areas, have been described as a potential target to improve immunotherapy (45). This is also relevant for the efficacy of radiotherapy, as potential systemic responses rely on an effective immune system (9). This is especially true for the eradication of metastases outside of the irradiated area, known as the abscopal effect (9). The mechanisms by which hypoxia influences the immune response have recently been reviewed in more detail (Barsoum and colleagues 2014, Noman and colleagues 2015, and Vito and colleagues 2020; refs. 25, 46, 47). A sustained reduction of hypoxia might be an effective strategy to lower immunosuppression and enhance the efficacy of radio- and immunotherapy in therapy-resistant hypoxic areas (13, 18).

Most treatments countering hypoxia-mediated radioresistance, such as accelerated radiotherapy with carbogen and nicotinamide (6), lead to temporary increases in tissue oxygen concentrations, which may be too short to result in a sustained effect on the local immune cell repertoire (48). Prolonging oxygenation can be achieved by reducing the oxygen consumption of tumor cells (49). Specifically, this can be achieved by inhibiting OXPHOS as has been demonstrated using [¹⁸F]fluoroazomycin arabinoside ([18F]FAZA)-PET imaging in an A375-R1 melanoma model 24 hours after administration of the inhibitor (50). Furthermore, increased proliferation of reoxygenated cells is not expected as these cells are inhibited in their capability to use the available oxygen. Pharmacologic inhibition of OXPHOS is already proposed as a therapeutic strategy for cancers in which this pathway is upregulated (Table 1), thereby blocking the tumor cells' primary source of ATP production. Inhibition of oxidative metabolism has also been suggested as a strategy to enhance the direct cytotoxic effects of radiotherapy (7, 8). As modulation of the TME can improve the efficacy of immune checkpoint blockade, radiotherapy, and its combination (51), inhibition of oxidative metabolism is a potential strategy to, by alleviating hypoxia, specifically sensitize hypoxic tumors to these treatment modalities. Overall and progression-free survival of patients with melanoma, following PD-1 blockade, more than doubled at 3 years, which was statistically significant, for tumors with a low oxidative metabolism and subsequent lesser hypoxia, posing OXPHOS as a potential target for improving immunotherapeutic response (44). Other studies also associate an increased mitochondrial metabolism with immunotherapy resistance or an immunosuppressive phenotype in melanoma (18, 52, 53). Several OXPHOS inhibitors have already been assessed for their anticancer properties and are making their way into the clinic (Table 1; Fig. 3). To select patients for treatment and to evaluate the antihypoxic effects of these OXPHOS inhibitors, functional hypoxia imaging is a useful tool that has already been used in some preclinical models and clinical trials.

Biguanides are a group of mitochondrial complex I–inhibiting molecules for treatment of type II diabetes mellitus (54). Metformin, one of the most commonly used biguanides, reduced hypoxia as measured by [18F]FAZA-PET imaging in LNCAP prostate cancer and HCT116 colorectal cancer xenografts or [¹⁸F]2-(4-((2-nitro-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)propan-1-ol ([18F]HX4)-PET imaging in A549 non–small cell lung cancer (NSCLC) and Colo205 colorectal cancer xenografts (8, 14, 55). Moreover, metformin induces a CD8⁺ T-cell–mediated antitumor effect by increasing the number of tumor-infiltrating lymphocytes and preventing T-cell exhaustion in the TME of several syngeneic tumor models (56). Also, metformin administered every 2 days intraperitoneally was able to potentiate PD-1 blockade in MC38 mouse colon cancer and B16 melanoma models by improving CD8⁺ T-cell function through upregulation of immunologic checkpoints, thereby reducing tumor growth (13). The closely related compound, phenformin, a more potent inhibitor of OXPHOS, shows a similar effect in Braf^V600E-induced/Pten-deficient melanoma by inducing CD8⁺ T-cell infiltration and inhibiting MDSC function (57). Metformin and phenformin also increase radiotherapy efficacy by alleviating tumor hypoxia in the CT26 colorectal cancer model, presumably by both directly sensitizing tumor cells to irradiation, and the aforementioned immune effects (58). Recruiting trials, investigating a combined use with radiotherapy or immune checkpoint inhibition are listed in Table 1. Although putative overall and disease-free survival benefits of metformin as a radio- and immunotherapy enhancer have been reported, the overall results so far remain inconsistent (59–61). Other antidiabetic drugs, like canagliflozin (a sodium-glucose cotransporter 2 inhibitor) and rosiglitazone (a peroxisome proliferator-activated receptors activator), both affecting OXPHOS (62, 63), are currently under clinical investigation. Canagliflozin has been suggested as a radiosensitizer in a hypoxic A549 NSCLC xenograft model (62), while the rosiglitazone trial is performed in combination with PD-1 blockade (Table 1). IM156 is another newly developed biguanide that inhibits mitochondrial complex I and impairs the growth of MYC-dependent lymphoma in vivo (64). IM156 is currently in clinical trials for advanced solid tumors (Table 1).

Statins, used for reducing cholesterol in cardiovascular disease through 3-hydroxy-3-methylglutaryl-coA reductase inhibition, are also inhibitors of HIF1α activation (65). However, the precise mechanisms involved remain unclear. Simvastatin has been identified as a potent inhibitor of mitochondrial complexes III and V, thereby potentially reducing hypoxia and explaining the inhibition of HIF1α (63). Moreover, simvastatin-mediated inhibition of tumor growth is attributed to HIF1α reduction and the suppression of TAM-meditated oxidative stress in B16 melanoma (65). The related atorvastatin is also likely an inhibitor of OXPHOS (66), and has been found to attenuate the activation of the HIF1 pathway under hypoxic culture conditions and enhance radiosensitivity in prostate cancer cells in vitro (67). This suggests that statins may alleviate tumor hypoxia via the inhibition of oxidative metabolism. Postdiagnosis statin use, especially atorvastatin and simvastatin, was associated with an increased survival of patients with lung and pancreatic cancer, as well as of patients with prostate cancer treated with radiotherapy (68–70). Clinical trials combining simvastatin and atorvastatin with radiotherapy for brain tumors have been conducted and the atorvastatin trial met criteria for continued accrual (Table 1).

Atovaquone, which is used to treat and prevent malaria, targets mitochondrial complex III and thereby collapses the malarial parasites mitochondrial membrane potential and energy supply (71). Atovaquone possesses the same inhibitory effect on mitochondrial complex III in MCF7 breast cancer cells (72). In pharynx and colorectal xenografts, administration of atovaquone daily via drinking water, decreased oxygen consumption, reduced tumor hypoxia, and sensitized tumors to radiotherapy (7). In addition, in a renal cell carcinoma xenograft model, the antitumor effect of the immunotherapeutic agent IFNα was potentiated by atovaquone (73). Furthermore, as a consequence of mitochondrial respiration inhibition, atovaquone has been shown to inhibit STAT3, a transcription factor involved in differentiation of immune suppressor cells, which may contribute to the reactivation of an immune response (74). Lately, atovaquone was investigated in clinical trials as a hypoxia modifier (Table 1).

IACS-010759 is a novel compound that potently and specifically inhibits mitochondrial complex I and can inhibit proliferation and induces apoptosis in tumor cells reliant on OXPHOS for their energetic needs (75). IACS-010759 is currently in clinical trials for advanced solid and hematopoietic malignancies (Table 1). Furthermore, daily IACS-010759 treatment showed to be effective in decreasing oxygen consumption and tumor hypoxia considerably in several xenograft models, which was illustrated by [18F]FAZA-PET imaging or pimonidazole staining (50, 53, 76, 77). Together, radiotherapy and IACS-010759 enhanced antitumor immunity and overcame PD-1 blockade resistance in a 344SQ murine lung cancer model, while triple therapy also promoted a systemic response and prolonged survival (18). Recently, another small-molecule inhibitor of mitochondrial complex I, ME-344 (78), has entered clinical trials in combination with bevacizumab, an antiangiogenic compound (Table 1). Results showed on target effects of ME-344, rendering it a promising OXPHOS inhibitor for combination therapies.

Nitric oxide (NO) has been suggested as a hypoxia modulator in cancer as it both decreases oxygen consumption (inhibiting OXPHOS) as well as induces vasodilatation (79, 80). NO inhibits mitochondrial complex IV and is administered via prodrugs, like nitroglycerine, via micellar systems, or other nanoscale carriers (80, 81). Daily in situ release of NO has proven to be effective in reducing tumor hypoxia, using [¹⁸F]fluoromisonidazole ([18F]FMISO)-PET imaging. It increases the levels of ROS formed by irradiation and improves the radiotherapeutic response in a Lewis lung carcinoma (LLC) model (81). In both B16 melanoma and 4T1 mammary carcinoma, hypoxia-induced PD-L1 expression was reduced by NO treatment, promoting T-cell–mediated lysis and inhibiting immune evasion (82). Moreover, in a prostate cancer xenograft model, NO reduced hypoxia and inhibited the abundance of TAMs (83), while NO increased the presence of tumor-infiltrating CD8⁺ and CD4⁺ T cells in murine HCA-1 hepatocellular carcinoma (80). The prodrug nitroglycerin, applied systemically, has been in clinical trials recently as a radiosensitizing agent (Table 1), but no survival benefit has been reported so far. A targeted approach using precision delivery of NO combined with radiotherapy yielded better results in preclinical models (81).

The most commonly used drug for estrogen receptor (ER)-positive breast cancer, tamoxifen, has been identified as a potent ER-independent inhibitor of mitochondrial complexes III–V (63), as well as mitochondrial complex I (84). By this mechanism, tamoxifen reduced oxygen consumption in an ER-negative MDA-MB231-LM2-4175 breast cancer xenograft (84). Targeted tamoxifen delivery reduced hypoxia 24 hours after administration, which increased the efficacy of photodynamic therapy in a mouse 4T1 breast cancer model, and suggest the same approach could be used to enhance radiotherapeutic response (85). In MCF7 breast cancer xenografts, however, the antiangiogenic and thrombogenic properties of tamoxifen induced hypoxia, underlining that its antihypoxic effect might be tumor dependent (86). In vitro, MCF7 cells were sensitized by tamoxifen to radiotherapy and tamoxifen is currently in clinical trials to elucidate whether the combination with radiotherapy can be beneficial for patient outcome (Table 1; ref. 87). The possible hypoxia-reducing properties of tamoxifen also render it a potential adjuvant for immune checkpoint blockade or other immune-stimulating therapies (88). Currently, a clinical trial (Table 1) is studying PD-L1 inhibition in patients with progressed breast cancer who are also treated with endocrine therapies, including tamoxifen, possibly enhancing the immunotherapeutic response.

Several other drugs, already approved for clinical use, have been accredited with the disruption of OXPHOS. An example is arsenic trioxide, used for the treatment of acute promyelocytic leukemia, which has also been suggested for the treatment of lung cancer, partly because of its potential radiosensitizing and immunostimulatory effects (89). In syngeneic transplantable liver tumor (TLT) and LLC models, treatment with arsenic trioxide reduced oxygen consumption and increased tissue oxygenation up to 2 hours after administration as measured by electron paramagnetic resonance oximetry, while also augmenting radiotherapy response in TLTs (90). Furthermore, arsenic trioxide treatment has been associated with the depletion of immunosuppressive cell types, like MDSCs and Tregs, while enhancing the antitumor activity of CD8⁺ T cells in several syngeneic mouse models, posing it as an immune activator (91, 92). Completed trials tested the efficacy of combining arsenic trioxide with radiotherapy, but no conclusive results have been reported yet (Table 1). Papaverine, an antispasmodic drug, was found to be a mitochondrial complex I inhibitor, causing reduced tumor hypoxia and increased radiotherapy response in A549 NSCLC and EO771 mammary cancer xenografts (93). Currently, papaverine is in phase I clinical trials as a radiosensitizing agent (Table 1). Finally, chlorpromazine, an antipsychotic dopamine antagonist with antiserotonergic and antihistaminergic properties, also inhibits OXPHOS (63) and is being investigated in the treatment of glioblastoma multiforme (Table 1).

Several trials have demonstrated that hypoxia-modifying agents can improve radiotherapy outcome, although these effects are overall modest (4). Clinical studies to validate whether hypoxia modifiers can enhance immunotherapy efficacy are needed and have only started recently. In this respect, measuring tumor hypoxia will be of utmost importance as most previous trials have not assessed patients on this basis and possibly overlooked beneficial effects (94). Monitoring hypoxic status can be used to select hypoxic patients, as tumors with pronounced hypoxia are more likely to respond to the proposed combination therapies. Furthermore, monitoring of hypoxia is important to validate whether the hypoxia-modifying treatment has the intended on-target effects. This is essential to better interpret the results of these clinical trials (95). Imaging, as opposed to taking a biopsy, is a preferred approach as it can assess the hypoxic status of the entire tumor, which is known to be very heterogeneous (95). Functional imaging also allows for noninvasive monitoring of hypoxic status over time, which can be helpful to determine follow-up changes in hypoxia during OXPHOS inhibition, both in clinical and preclinical settings. Several methods for imaging hypoxia have made their way into the clinic already (96). The most commonly applied methods to date involve PET using redox-sensitive probes, of which nitroimidazoles are currently the most used class. Of these, [18F]HX4-PET (14, 55), [18F]FAZA-PET (50, 76), and [18F]FMISO-PET (81) have been used to asses hypoxia-alleviating effects of OXPHOS inhibitors in preclinical models, while [18F]FAZA-PET and [18F]HX4-PET are also used in some clinical trials (Table 1). Alternatively, targeting an endogenous hypoxia marker could be advantageous as it visualizes tumor cells that adapted to the harsh hypoxic TME. A loss of signal in this case would represent a durable immune-permissive reprogramming of the TME. In this respect, carbonic anhydrase IX is a promising marker and is currently under preclinical investigation as an imaging target (97).

Several preclinical studies have shown that the inhibition of OXPHOS can structurally alleviate hypoxia by repressing oxygen consumption, thereby increasing the efficacy of both radio- and immunotherapy and its combination. Although these results are very promising in preclinical models, hypoxia-alleviating effects and subsequent survival benefits have yet to be proven in the clinic. OXPHOS inhibition to alleviate hypoxia will most likely only benefit patients with tumor hypoxia because of an enhanced oxygen consumption. Furthermore, the immunosuppressive action caused by acidosis of the TME as a consequence of glycolytic metabolism will not be eliminated by OXPHOS inhibition, and may even be intensified by inhibiting mitochondrial metabolism. Another point of discussion is the influence of tumor mitochondrial metabolism on immunotherapy efficacy, as upregulated OXPHOS has been described to be associated with immunotherapy response (98), as well as resistance (18, 44, 52, 53). A possible explanation could be that oxidative metabolism is not in all cases correlated with tumor hypoxia, once more emphasizing the complexity of cancer biology and the importance of personalized medicine.

Several OXPHOS inhibiting compounds have made their way into clinical testing. Commonly used drugs, such as biguanides and statins, have shown a beneficial effect for patients with cancer in retrospective studies. They may be easy to implement in clinical practice, as are other commonly used drugs, like tamoxifen, NO, and arsenic trioxide. However, as these so-called “dirty drugs” are not very selective, they might have significant off-target effects (99) and it could be hard to pinpoint whether the reason for alleviated immunosuppression is a sustained increase in tissue oxygenation. Furthermore, clinical trials focused on the validation of hypoxia modification by these drugs are still needed. To date, only a few clinical trials have incorporated functional imaging to confirm antihypoxic effects and fewer use imaging for patient selection. More potent and specific mitochondrial inhibitors, like atovaquone and IACS-010759, have shown to effectively alleviate hypoxia and increased the efficacy of immunotherapy in preclinical studies, suggesting this is because of a sustained increase in tissue oxygenation (18, 73). These drugs might be better candidates for a targeted antihypoxia approach and together with appropriate imaging and response monitoring lead the way to increased clinical benefit in selected patients when combined with radiotherapy and immunotherapy.

This review describes how the inhibition of OXPHOS may be a promising strategy to structurally alleviate hypoxia and reactivate an antitumor immune response. Several OXPHOS inhibitors have shown promising preclinical results and have proceeded into clinical trials. Meticulous patient selection and hypoxia monitoring, however, are crucial to validate their hypoxia-modifying effects, these are absent in many trials. Such future clinical studies should show the value of OXPHOS-inhibiting compounds in improving the efficacy of radio- and immunotherapy combinations.

No potential conflicts of interest were disclosed.

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