Glucose consumption is one MCP granting cancer cell survival, restricting T-cell activation and promoting resistance to adoptive T-cell therapy (96). Both anaerobic and aerobic glycolysis are amenable to therapeutic intervention. Myeloma cells exhibit increased activity in glucose transporters of GLUT family like GLUT1, GLUT4, GLUT8 and GLUT11, and in glycolytic enzymes like HK (97). Interestingly, myeloma cells rely on the insulin-responsive glucose transporter GLUT4 for basal glucose consumption, maintenance of Mcl-1 expression, growth, and survival. The FDA-approved HIV protease inhibitor ritonavir acts as a GLUT4 off-target inhibitor abrogating both glucose uptake and proliferation on KMS11 and L363 myeloma cells (97). Interestingly, some myeloma cells can survive to glucose deprivation and/or ritonavir treatment because they switch to OXPHOS to support their energetic needs. Metformin is an FDA approved antidiabetic medication that targets mitochondrial complex 1. The combination of ritonavir and metformin in vitro induces the apoptosis of myeloma cell lines and human primary myeloma cells, and the same combination has shown antimyeloma activity in vivo in a xenograft myeloma mouse model (98). Inhibition of AKT and mTORC1 phosphorylation in combination with Mcl-1 down-regulation have been advocated as key mechanisms (98), but it is also possible that a major contribution comes from the energetic crash induced by the ritonavir and metformin combination. Dichloroacetate, an inhibitor of anaerobic glycolysis, has been reported to increase myeloma cell sensitivity to bortezomib (99), and Zub et al. have confirmed that inhibition of aerobic glycolysis increases sensitivity of myeloma cells to melphalan (100).

Pyruvate kinase isoform M2 (PKM2) catalyzes the final rate-limiting glycolytic reaction and plays a key role in cancer cell metabolism and immune responses. Furthermore, PKM2 dimers can translocate into the nucleus and stimulate HIF-1α-dependent transcriptional activity. Intriguingly, PD-L1 is up-regulated by PKM2 and HIF-1α in macrophages, DC, T cells and tumor cells. The small molecule TEPP-46 partially relieves the immune suppressive TME contexture by down-regulating PD-L1 expression via PKM2 inhibition (101). Additional ICP/ICP-L blockade (i.e., TIM3, LAG-3 etc) can further improve TEPP-46 activity and ameliorate antitumor immune responses.

ICP/ICP-L blockade itself can down-regulate glycolytic fluxes. Anti- ICP/ICP-L mAbs in combination with glycolytic inhibitors can restrain the extensive glucose uptake and utilization by tumor cells and redirect this energy resource towards effector T cells to sustain their reactivity and expansion (94). The increased glycolytic flux in tumors is controlled by AKT/mTOR pathway that up-regulates the glucose uptake via specific GLUT transporters and glycolytic enzymes (67). Anti-PD-L1 mAbs reduce glycolysis in cancer cells by inhibiting Akt/mTOR signaling making glucose also available to T cells to fuel their metabolic needs and mount antitumor immune responses (67, 102, 94). A plus for ICP/ICP-L mAbs compared with traditional glycolytic inhibitors (i.e, fasentin, lonidamine, phosphonoacetohydroxamate, galloflavin, etc) is that they target more specifically tumor cells. The combination of ICP/ICP-L inhibitors with sub-therapeutic concentrations of glycolytic inhibitors (to prevent side effects) can have synergistic effect on the antitumor activity of T cells in the TME.

Akt/mTOR inhibitors also can be combined with ICP/ICP-L mAbs to promote a fairer glucose redistribution in the TME. Interestingly, it has been reported a tight association between ICP-L expression and mTOR activity in cancer cells (103) suggesting that their concurrent inhibition can result in two therapeutic effects: 1) inhibition of tumor cell growth by interfering with the pro-survival pathways driven by Akt/mTOR signaling; 2) suppression of glucose over-utilization by tumor cells and recovery of antitumor immune functions, including Vγ9Vδ2 T cells. Lastly, the combination of ICP/ICP-L blockade and Akt/mTOR inhibition could also increase the sensitivity of myeloma cells to conventional chemotherapy opening the way to triple combination treatments.

The TCA cycle and OXPHOS are mitochondrial pathways acting downstream to aerobic glycolysis. The TCA cycle benefits also from the anaplerotic fluxes of FAO and glutaminolysis. These pathways are able to provide long-term energy supply and provide building blocks for proliferating cells and immune cells undergoing functional differentiation.

Myeloma cells rely on the TCA cycle, including its anaplerotic fluxes, and OXPHOS to meet their energy requirements (14, 15, 104). Disruption of these energy sources has a strong impact on tumor cell survival and proliferation. Mitochondria have emerged as an intriguing target for anticancer drugs, inherent to the great majority of tumors. Drugs that target mitochondria and exert anticancer activity have been termed 'mitocans' to outline their ability to induce mitochondria destabilization (105). The TCA inhibitors phosphonoethyl ester of succinyl phosphonate (an α-ketoglutarate dehydrogenase inhibitor) and AGI-6780 (an isocitrate dehydrogenase – IDH - inhibitor), the glutaminolysis inhibitor BPTES, and the electron transport chain (ETC) inhibitor α-tocopheryl succinate (complex II inhibitor) are FDA-approved agents under clinical evaluation in several tumors and potentially transferable to MM. Targeting the mitochondrial metabolism has also already been explored in MM patients in combination with proteasome inhibitors. The combination of carfilzomib and the IDH2 inhibitor AGI-6780 is lethal to primary myeloma cells in vitro because the TCA cycle is disrupted and ATP is no more available. Since IDH2 is activated by the NAD⁺-dependent deacetylation mediated by sirtuin-3, the use of NAD⁺-generating enzyme nicotinamide phosphoribosyltransferase (NAMPT) inhibitors is another potential tool to improve the efficacy of carfilzomib and overcome the development of carfilzomib resistance (106).

Mitocans are not exempted from detrimental effects because mitochondrial biogenesis is also required by effector T cells to mount effective antitumor immune responses. Tumor-infiltrating lymphocytes (TIL) are characterized by low mitochondrial mass and metabolic flux due to the Akt-mediated inhibition of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), a transcription factor that promotes mitochondrial biogenesis. Restoring the activity of PGC1α corrects the defective mitochondrial mass and increases the energy supply for TIL, promoting their sustained activation and effector functions (107). Mitochondrial biogenesis is emerging as critical issue also to improve the efficacy and duration of chimeric antigen receptor (CAR) redirected T cells (CAR-T cells). The costimulatory domain 4-1BB has replaced CD28 in the architecture of second-generation CAR-T cells because it promotes the outgrowth of CD8⁺ memory T cells, which have significantly better respiratory capacity, increased FAO and enhanced mitochondrial biogenesis (108). Very recently, Van Bruggen et al. have reported that the clinical efficacy of CAR-T cells in CLL is dependent on their mitochondrial mass which can be regarded as an indirect evidence of their capacity to successfully compete with energy sources in the TME (109).

Unfortunately, mitocans, like other metabolic correctors, are penalized by the lack of specificity leading to contrasting effects on tumor cells and immune effector cells in the TME. It is a matter of debate whether the conjugation of mitocans with tumor-selective ligands and/or the conjugation of mitochondrial reactivating agents (e.g. PGC1α inducers) with T-lymphocytes-targeting ligands may overcome this impasse. Conjugation of mitochondrial activators with pAgs specifically recognized by Vγ9Vδ2 T cells may represent a promising strategy to improve the selectivity of metabolic correctors.

The hypoxic TME in MM BM leads to the constitutive up-regulation of HIF-1α, a strong transcriptional inducer of GLUT and glycolytic enzymes. Therefore, HIF-1α inhibition can decrease glucose utilization by myeloma cells making glucose available to immune effector cells including Vγ9Vδ2 T cells. Unfortunately, HIF-1 inhibitors have failed clinical expectations so far, probably because they lack specificity (110). Oxygen consumption by ETC in tumor cells is also under investigation as a targetable MCP. Hypoxia promotes ICP/ICP-L expression via HIF-1α activation and other mechanisms and preliminary data indicate that ETC inhibitors attenuates hypoxia in the BM of MM patients and increase the efficacy of anti- ICP/ICP-L mAbs (107).

These data confirm that the ICP/ICP-L circuitry is an attractive candidate to combine with HIF-1 inhibitors. Hypoxia increases the number of PD-L1⁺ MDSC and Tregs in the TME, and targeting hypoxia-driven MCP can prevent the accumulation of these immune suppressive cells (111). The interactions between hypoxia-driven MCP and ICP/ICP-L can also be mediated by exosomes. Interestingly, it has been reported in oral squamous cell carcinoma that exosomes derived from normoxic tumors expand immunocompetent γδ T cells (mainly represented by Vγ9Vδ2 T cells), whereas exosomes derived from hypoxic tumors expand PD-L1⁺ MDSC that recruit PD1+ anergic γδ T cells (112). This work demonstrates that oxygen pressure in the TME regulates the induction of anti- or pro-tumoral γδ T cells by modulating the release of exosomes by tumor cells.

HIF-1β, a transcription factor steadily translocated in the nucleus to activate the transcriptional program mediated by HIF-1α in hypoxia, is a strong inducer of ABCA1 which is highly expressed in hypoxic endothelial cells (113) and macrophages (114). BMSC of MM patients, deep-rooted in a constitutively hypoxic TME, display high levels of ABCA1 and extrude huge IPP amounts in the TME (49). We are currently investigating whether these unnatural IPP concentrations (in the micromolar range) determine a functional exhaustion of Vγ9Vδ2 T cells by increasing, in the absence of appropriate co-stimulatory signals, the expression of multiple ICP (PD-1, TIM3, LAG-3) (47, 5, 49, 7). We have initiated to investigate whether HIF-1 inhibitors can rescue the immune competence of Vγ9Vδ2 T cells in the TME of MM patients by decreasing ABCA1 expression and extracellular IPP release from BMSC. As said above, metabolic corrections are often double-edged approaches. In this case, decreasing the HIF-1/ABCA1 axis in BMSC may limit the long-term and uncontrolled exposure of BM Vγ9Vδ2 T cells to supra-physiological IPP concentrations and relieve their anergy and senescence, but the down-regulation of IPP production and release by DC may concurrently restrain the activation of Vγ9Vδ2 T cells. In-depth investigation of different expression of cell surface antigens by BMSC vs DC, coupled with medicinal chemistry approaches to conjugate HIF-1 inhibitors with ligands specific for immune suppressive cells, are current under investigation to successfully introduce HIF-1 inhibitors in the clinical setting.

Interestingly, metformin, an anti-diabetic drug that inhibits OXPHO, reduces oxygen consumption in the TME and improves oxygen concentrations (115, 116). The reduced hypoxia relieves several immune suppressive features and increases the efficacy of PD-1 blockade. Metformin alone does not have a significant antitumor activity, but in combination with anti-PD-1 mAb locally increases the number and function of CD8⁺ cells with antitumor activity (i.e. producing TNF-α and IFN-γ) (107). It is currently unknown the impact of metformin or other hypoxia-targeted treatments on Vγ9Vδ2 T cells under normoxic or hypoxic conditions. Nevertheless, since Vγ9Vδ2 T cells are highly sensitive to the metabolic TME alterations, including hypoxia, it is very likely that remodeling the hypoxic tone in the TME of MM patients is immunologically beneficial also to Vγ9Vδ2 T cells. Besides relieving hypoxia, metformin can be particularly useful in MM patients because it consistently decreases IL-6R expression in myeloma cells (117). IL-6 can promote, in combination with TGF-β and other cytokines highly abundant in the TME of MM patients, the differentiation of IL-17 producing γδ T cells which support tumor cell progression by promoting angiogenesis (118).

Lastly, hypoxia is considered a strong protumoral metabolic change in the TME because it favours the emergence of MDR (119). However, recent data suggest that hypoxia can be exploited to induce the selective activation of prodrugs in the TME. For instance, evofosfamide is a prodrug of the DNA alkylator bromo-isophosphoramide that is activated under hypoxic conditions. environment. Evofosfamide in combination with bortezomib and dexamethasone has shown a safe profile and clinical efficacy in patients with relapsed MM who have failed standard treatments (120).

ICP/ICP-L blockade can also be combined with Mev pathway manipulation to improve antitumor immune responses. Promising data have been generated in murine cancer models where inhibition of acetyl citrate lyase (ACLY), the main generator of cytosolic acetylCoA used in cholesterol synthesis, enhances the pro-immunogenic effects of chemotherapy and the efficacy of antitumor T cells. The inhibition of acetylCoA acetyltransferase-1 (ACAT1) that promotes a feedback inhibition of Mev pathway by preventing the esterification of cholesterol, produces similar effects (121, 122). These two metabolic modifications can be used in association with ICP/ICP-L blockade to boost the antimyeloma activity of Vγ9Vδ2 T cells.

Modulation of the Mev pathway can take advantage of the hypoxic TME of MM patients. HMGCR is down-regulated by hypoxia. Direct inhibitors of HMGCR like statins or down-stream FPPS inhibitors like NBP are more cytotoxic against cancer cell lines under hypoxic than normoxic conditions (123). One possible explanation is that the deeper Mev pathway inhibition leads to a drastic intracellular deprivation of cholesterol and isoprenoids that myeloma cells cannot tolerate under hypoxic conditions. Another mechanism can be the BTN3A1 conformational changes induced by NBP-induced intracellular IPP accumulation cells that can increase myeloma cell recognition by Vγ9Vδ2 T cells. The first mechanism may explain why statins and compounds like brutieridin and melitidin, that incorportate the HMG moiety (124), may have potent antitumor effects (125-127) even if they do not induce IPP accumulation and stimulate Vγ9Vδ2 T cells (5, 47, 128). Since lipohilic statins inhibit HIF-1α activity in solid tumors, it is worth investigating whether these drugs can also suppress PD-1L transcription. So far, no data are available about the capacity of statins to inhibit HIF-1α activation in tumor-infiltrating lymphocytes and/or Vγ9Vδ2 T cells. Future research in this field may help to understand whether the combination of lipophilic statins and ICP/ICP-L blockade may enhance antitumor immune responses in the TME of MM patients and eliminate residual MDR+ myeloma cells nested in the hypoxic BM niches.

Beside the Mev pathway, other lipid metabolic pathways are important mediators of antitumor immune responses and therefore potentially druggable MCP. Any modifications in cellular lipid metabolism significantly affects T-cell fate and function (129). Indeed, the activation-induced proliferation and differentiation of effector T cells is supported by FAS, whereas the development of CD8⁺ T cell memory cells requires FAO (130, 131). As a consequence, long-lasting memory-like T-cells rely preferentially on mitochondrial OXPHOS and FAO for meeting their energetic demands (130, 132).

Enhanced FA utilization can be an important metabolic opportunity for exhausted T cells that are highly dependent on FAO to support their energetic needs. In the presence of glucose deprivation, FAO becomes the main fueling pathway to preserve the effector functions of CD8⁺ T cells in the TME. Fenofibrate, a PPAR-α activator with the capacity to increase FAO, sustains the immune competence of CD8⁺ TIL and delays their functional exhaustion irrespective of PD-1 expression. Interestingly, fenofibrate has been reported to synergize with anti-PD-1 blockade in melanoma (133). Similarly, chemical agents enhancing OXPHOS in CD8⁺ TIL increase the efficacy of anti-PD1 treatments in colon cancer models (134). These data suggest that increasing FAO and OXPHOS in TIL, in combination with anti-ICP/ICP-L treatment, can represent a significant advance in preventing the functional exhaustion of TIL in the TME. Unfortunately, increasing FAO in T cells may induce unwanted effects on the delicate balance between activation and inhibition of immune cells. FAO is also the main energetic pathway used by Treg cells and MDSC (135-137) to differentiate and exert their immune suppressive activity on T cells. As a consequence, FAO inhibition can prevent Treg accumulation, but it impairs effector T-cell functions; contrariwise, FAO up-regulation can improve T-cell functions, but it increases immunosuppressive functions by Treg cells and MDSC (135).

Lipid metabolism can play a special role in BM-located malignancies like MM. The proportion of BM adipocytes increases with age and therefore it is not surprising that these cells are particularly abundant in the BM of MM patients which is a disease of the older population. Adipocytes in the TME of MM patients can provide FA and triglycerides to myeloma cells (138) that are strategically equipped with appropriate receptors like CD36 and the FA Transporter Proteins (FATPs) to internalize these invaluable energy sources (139, 140). Etomoxir has been used to deprive myeloma cells of FA via inhibition of carnitine palimtoyl transferase 1 (CPT1), the pace-maker enzyme in FAO, yielding to a significant suppression of myeloma cell proliferation (140). Myeloma cells can also produce FA via FAS and this pathway is instrumental to metabolically support their survival and expansion. Interestingly, the combination of etomoxir with orlistat (a FAS inhibitor) synergistically suppresses myeloma cell growth and enhances the sensitivity to bortezomib treatment (141). Although promising, these findings should be regarded with caution because the results have been generated using myeloma cell cultures taken out of the immune suppressive TME.

Very little data are available about lipid metabolism in Vγ9Vδ2 T cells. Recently, it has been shown that IL-21 reduces aerobic glycolysis and increases FAO in T cells. This metabolic reset is paralleled by increased mitochondrial biogenesis to generate long-lasting memory T cells with low PD-1 expression (142). Interestingly, normal Vγ9Vδ2 T cells have enhanced antitumor activity if expanded in the presence of IL-21 and IL-2, whereas Vγ9Vδ2 T cells from cancer patients have defective reactivity to IL-21 stimulation (143). Vγ9Vδ2 T cells from patients with acute myeloid leukemia display lower expression of IL-21R and require higher levels of IL-21 for their expansion. These cells also express significantly higher TIM-3 levels than healthy Vγ9Vδ2 T cells and up-regulate TIM-3 after IL-21 stimulation. Blocking TIM-3 increases the proliferation and reactivity of Vγ9Vδ2 T cells to IL-21 stimulation (144). These data indicate the existence of druggable interconnections between ICP/ICP-L and FA metabolism mediated by cytokines like IL-21. Preliminary data in our laboratory indicate that anergic Vγ9Vδ2 T cells isolated from the TME of MM patients may up-regulate multiple ICP when stimulate by ZA-treated DC in the presence of IL-2. Altogether, these data confirm the multifaceted reactivity of Vγ9Vδ2 T cells embedded in the TME and how their functional plasticity dangerously allows them to switch from antitumoral to protumoral functions (145).

IDO inhibition is under investigation as a druggable MCP to improve the efficacy of ICP/ICP-L inhibition. IDO inhibitors can rescue dysfunctional or exhausted T cells by raising tryptophan levels in the TME. L-methyl-tryptophan (1-MT) (146) and INCB024360 (147) have been shown to improve the immune responses of tumor-specific T cells in mouse models. Following these initial observations, indoximod (IDO1 and IDO2 inhibitor), navoximod (IDO1 inhibitor, also known as GDC-0919 and NLG919) and the dual IDO1–TDO inhibitors HTI-1090 (IDO1-TDO inhibitor) also known as SHR9146) and DN1406131 have been developed and investigated as single agents or in combination with ICP/ICP-L blockade (148). Holmgaard et al. have shown that IDO inhibition in combination with CTLA-4, PD-1/PD-L1, and GITR blockade has synergistic activity on tumor cell growth and improves the survival in different tumor models (149). Based on these promising results, clinical trials are currently underway to investigate the combination of anti–CTLA-4 mAb and IDO inhibition in patients with melanoma. The very potent and highly selective IDO1 inhibitor epacadostat (INCB024360) is under clinical investigation as single agent in various advanced-stage malignancies and under consideration in combination with anti-ICP/ICP-L mAbs.

The combination of IDO inhibitors and ICP/ICP-L blockade is also worth investigating in in MM given the well-known role played by in the TME of these patients. A preclinical rationale has been provided by An et al. who have shown that anti-PD-L1 mAb in combination with IDO inhibition can overcome the osteoclast-induced suppression of T-cell mediated antimyeloma immune responses (150). IDO inhibition can also alleviate Vγ9Vδ2 T-cell immunosuppression induced by the overproduction of kynurenines by myeloma cells and MDSC in the TME.

Arginine deprivation is another major MCP restraining antitumor immune responses mediated by T cells and NK cells in the TME (151). Arginine supply and/or prevention of arginine degradation via Arg1 inhibition are feasible strategies to reinstate antitumor immune responses in the TME. As expected, arginine supplementation revives the cytotoxic functions of T cells and NK cells, and, in combination with anti-PD-L1 mAb, enhances antitumor immune responses and prolongs the survival of tumor-bearing mice (152). CB-1158, a potent and orally-bioavailable small-molecule Arg1 inhibitor, has shown efficacy in murine syngeneic tumor models (153) as single agent or in triple combination with adoptive T-cell or NK-cell therapy, adoptive NK cell therapy, and anti-PD-L1 mAb. The combination of CB-1158 and anti-PD-L1 mAb is currently under clinical investigation in patients with advanced/metastatic solid tumors (NCT02903914, INCB 01158-10) (154).

Interestingly, arginine metabolism can also be modulated in the TME by targeting its conversion into citrulline. In the presence of arginine deprivation, normal tissues can increase citrulline uptake and up-regulate the expression of argininosuccinate synthetase 1 (ASS1) which converts citrulline into arginine. This auxotrophy mechanism can be defective in tumor cells that are unable to up-regulate or lack ASS1 (155). Pegylated arginine deiminase (ADI-PEG 20) has been develop to convert arginine into citrulline and ammonia, making ASS1-deficient tumor cells unable to use citrulline to generate arginine and meet their metabolic needs (156). ADI-PEG 20 has shown antitumor activity in ASS1-deficient tumor cells from patients with breast cancer (157), small-cell lung cancer (158) and acute myeloid leukemia (159). Interestingly, T cells can increase citrulline uptake and up-regulate ASS1 expression under low arginine conditions (160-162). Thus, the conversion of arginine into citrulline by ADI-PEG 20 may have different effects on T cells compared with arginine depletion induced by Arg1. Indeed, Brin et al. have shown that peripheral blood T cells from healthy donors can be fully activated in the presence of ADI-PEG 20 avoiding PD-1 up-regulation and the differentiation into Treg cells. In murine syngeneic tumor models, ADI-PEG 20 has been reported to promote the recruitment of T cells in the TME and improve the efficacy of anti-PD-L1 treatment (163).

Targeting Arg1 can also be effective in MM. We have shown that PD-L1+ MDSC are significantly expanded in the TME of MGUS and MM patients, irrespective of the percentage of TME-infiltrating myeloma cells (5). Romano A. et al. have recently shown that Arg-1 is highly expressed by MDSC of MM patients (164). Arg-1 inhibition improves the in vitro antimyeloma activity of bortezomib and lenalidomide and this combination is currently tested in animal models to determine safety and efficacy in vivo. We are currently investigating whether Arg-1 up-regulation and arginine deprivation are involved in the dysfunction of BM Vγ9Vδ2 T cells in MGUS and MM patients.

Borrello’s group has pursued a different strategy to target the immune suppressive functions of MDSC in the TME of MM patient (165). They have shown that sildenafil down-regulates Arg1 and nitric oxide synthase–2 (NOS2) expression, thereby reducing the suppressor function of MDSC and ameliorating antimyeloma immune responses mediated by T cells. In the clinical setting, they have shown that tadalafil, another phosphodiesterase-5 (PDE5) inhibitor, attenuates the suppressor functions of MDSC and allows the achievement of measurable antimyeloma immune responses (166). When added to chemotherapy, this approach has improved the clinical outcome of end-stage relapsed/refractory MM. We have investigated the effects of PDE5 inhibitor sildenafil on pAg-reactivity of BM Vγ9Vδ2 T cells from MM patients and failed to show any functional recovery, further confirming the deeper impairment of these cells compared with conventional T cells (5).

MDSC also express high levels of NOS2, which convert arginine into NO. NO can suppresses T-cell function either by S‑nitrosylation of critical cysteine protein residues or by regulation of guanylyl cyclase and cyclic GMP-dependent kinases (167). Sharing the same substrate, NOS2 and Arg-1 are in dynamic competition with the former having much higher affinity for arginine, but much slower catalytic activity. In the presence of severe arginine depletion, NOS2 switches from arginine degradation to the generation of reactive oxygen species (ROS) and reactive nitrogen species which are immune suppressor metabolites. These data suggest that the combination of Arg1 and NOS2 inhibitors should ensure the most effective MDSC inhibition. NCX‑4016 (nitroaspirin), a small molecule developed to test this hypothesis, has been shown to correct the immune dysfunction and promotes tumor eradication after cancer vaccination in tumor-bearing mice (168).

Targeting NOS2 could be particularly rewarding to rescue the immunocompetence of Vγ9Vδ2 T cells. Douguet et al. have shown that a significant proportion of γδ T cells infiltrating mouse and human melanoma express NOS2. Experimental data in the murine melanoma model indicate that NOS2 can be involved in the generation and expansion of IL-17-producers tumor-promoting γδ T cells which, in turn, can promote the recruitment and accumulation of MDSC in the TME (169). It is currently unknown whether NOS2 is also expressed by BM Vγ9Vδ2 T cells from other cancers, but the combination of NOS2 inhibitors and ICP/ICP-L blockade sounds very interesting to recruit immune effector cells, including Vγ9Vδ2 T cells, in the TME of MGUS and MM patients.

Glutamine metabolism is critical for tumor cell survival and therefore another attractive MCP to target for therapeutic interventions. The whole pathway has been considered and several compounds developed, from glutamine analogs to inhibitors of glutamine transporters or inhibitors of glutamate conversion to α‑ketoglutarate (170). Some of these compounds have moved on to clinical trials as single agents or in association with anti-ICP/ICP-L mAbs (148).

The importance of glutamine metabolism in myeloma cells have arisen from the clinical observation that a significant proportion of MM patients at diagnosis are affected by hyperammonemia and encephalopathy in the absence of liver dysfunction. Subsequently, it has been shown that human myeloma cell lines and primary myeloma cells, lacking a detectable expression of glutamine synthetase (GS), rely on the uptake of extracellular glutamine, mainly via the ASCT2 glutamine transporter, for their growth and survival. Bolzoni et al have shown that blocking glutamine uptake significantly affects the viability of myeloma cells and their sensitivity to bortezomib and other drugs. They have also shown that stable ASCT2 downregulation by a lentiviral approach inhibits the growth of human myeloma cell in vitro and in a murine model. Similar data have been reported using the combination of glutaminase (GLS) inhibitor CB-839 alone or in combination with pomalidomide (171, 172) or glutamine deprivation in the presence of venetoclax (173).

Targeting glutamine metabolism in myeloma cells may have many-sided effects on T cells in the TME. Glutamine uptake and glutaminolysis are up-regulated in naïve T cells after activation to support their proliferation (62). Following activation, glutamine levels can regulate the functional differentiation of specific CD4+ and CD8+ subsets. In the presence of adequate glutamine supply, effector T cells can shift from glycolysis to OXPHOS to generate central memory-like cells with enhanced survival and antitumor activity (71). In the presence of glutamine deprivation, including ASCT2 deletion, T-cell activation results in the differentiation into Foxp3+ Tregs cells that further suppress T-cell activity (70). More recently, it has been shown that inhibition of glutaminolysis at the level of GLS can have different effects on T-cell differentiation compared to depletion of extracellular sources. Johnson et al have shown that GLS promotes the functional differentiation of Th17 cells in spite of Th1 cells. Transient GLS inhibition leads to increased Th1 differentiation and the generation of CD8+ CTL effector functions (174). It is currently unknown whether glutamine metabolism, either as deprivation of external source or as impaired GLS activity, may impact on the functional differentiation of Vγ9Vδ2 T-cell subsets. Interestingly, IL-17-producing γδ T cells have been reported to be generated in the TME of tumor-bearing mouse and to behave as tumor-promoting cells (175). We are currently investigating whether the glutamine deprivation induced by myeloma cells in the TME in the presence of IL-6 and TGF-β promotes the generation of IL-17-producing Vγ9Vδ2 T cells as in tumor-bearing mice.

Given the strong impact that manipulation of glutamine metabolism may have on antitumor immune responses, it is not suprising that clinical trials are under investigation in combination with anti-ICP/ICP-L mAbs. Trigriluzole (BHV-4157), a compound that reduces extracellular glutamate levels, is currently tested in combination with nivolumab or pembrolizumab in metastatic or unresectable solid tumors or lymphoma (NCT03229278), while CB-839 (glutaminase 1 inhibitor) is tested in combination with nivolumab in advanced- stage clear cell renal carcinoma, melanoma and non small cell lung cancer (NCT02771626) (148).

Ado is an important MCP in the hypoxic BM niche where residual myeloma cells can survive chemotherapy and immunotherapy (176). Ado, through its receptor A2AR, can up-regulate PD-1 expression on CD8+ and Treg cells (177) and the dual blockade of PD-1 and A2AR synergistically increases the cytotoxic potential of CD8+ cells and controls tumor growth (178). Combination of A2AR inhibition with TIM-3 or CTLA-4 blockade has also been shown to be effective in enhancing antitumor immune responses in a variety of syngeneic tumor models (179).

The role of Ado in the immune suppressive TME of MM patients is also under investigation (180). Yang R et al. have reported increased Ado concentration in the BM plasma from MM patients compared with healthy controls (181). CD39 expression by myeloma cells coupled with CD73 expression by BMSC and other bystander cells are responsible for the generation of immunosuppressive extracellular Ado concentrations in the TME leading to A2AR-mediated T-cell inhibition. The CD39 inhibitor POM1 inhibits Ado generation in myeloma cell/BMSC co-cultures and restores T-cell proliferation in vitro. Interestingly, high CD39 expression in the TME of MM patients confers a poor clinical outcome further supporting the role Ado in the immunosuppressive TME of these patients (181). Richles RJ et al. have shown that A2AR agonists in combination with PDE inhibitors act synergistically in triple combination with glucocorticoid to suppress the proliferation of myeloma cells in vitro (182).

Extracellular Ado in the TME of MM patients can also be generated from nicotinamide dinucleotide (NAD) by the concerted action of CD38, CD203a and CD73 (183). CD38 is a multifunctional ecto-enzyme acting also as a receptor and adhesion molecule in the TME of MM patients. Being expressed by myeloma cells and several other lymphoid and myeloid cells in the TME, CD38 is strategically located as a target intersection to modulate tumor-host interactions (184). Several anti-CD38 mAb like daratumumab, isatuximab, and MOR202 have been developed and clinically used in MM patients as single agents or in combination with steroids and other drugs (185). Although several cell subsets express both CD38 and ICP/ICP-L in the TME of MM patients (including myeloma cells, immune suppressor cells and immune effector cells), very little data are available about the cascade of reciprocal interactions eventually triggered by anti-CD38 or anti-ICP/ICP-L mAbs. Chen et al. have reported in lung cancer and animal models that acquired resistance to anti-PD-1/PD-L1 mAbs is mediated by CD38 upregulation and shown that concurrent CD38 and PD-1/PD-L1 inhibition improves antitumor immune responses (186).

The role of Ado pathway in γδ T-cell functions is much less recognized. Liang D. et al. have addressed this issue in an experimental autoimmune uveitis mouse model (187). They have shown that the CD73 and A2AR expression on γδ T cells are dependent on their activation status and modulate immune responses mediated by Th17 and Treg cells by fine-tuning extracellular Ado concentration (188, 189). CD73 and CD39 expression has been correlated with the acquisition of suppressor functions in Vγ9Vδ2 T cells after exposure to pAg in the presence of inappropriate cytokines (190, 191). CD39 upregulation has been reported to dephosphorylate self and microbial pAgs restraining the reactivity of Vγ9Vδ2 T cells (192).