Mitochondrial metabolism: Inducer or therapeutic target in tumor immune-resistance?

Mitochondria have been considered for a long time only as the principal source of building blocks and energy upon aerobic conditions. Recently they emerged as key players in cell proliferation, invasion and resistance to therapy. The most aggressive tumors are able to evade the immune-surveillance. Alterations in the mitochondria metabolism either in cancer cells or in host immune system cells are involved in such tumor-induced immune-suppression. This review will focus on the main mitochondrial dysfunctions in tumor and immune cell populations determining immune-resistance, and on the therapies that may target mitochondrial metabolism and restore a powerful anti-tumor immune-activity.

Molecular dynamics of targeting CD38 in multiple myeloma.

Multiple functions of CD38 need exploring to expand clinical application of anti-CD38 antibodies in multiple myeloma (MM). We investigated membrane dynamics of MM cells and subsequent events when CD38 is targeted by therapeutic antibodies. Human MM cells (BF01) were co-cultured in vitro with therapeutic antibody (or control immunoglobulin G) and analysed using gene expression profiling. Microvesicles from antibody-exposed cells were analysed for differential gene and microRNA (miRNA) expression, and for phenotypic characterisation. Exposure of BF01 cells to anti-CD38 antibody resulted in CD38 membrane redistribution, upregulation of metabolism-related genes and downregulation of genes involved in cell cycle processes. Microvesicles derived from antibody-exposed cells showed increased CD73 and CD39 expression, presence of programmed death-ligand 1 and significant up-/down-modulation of miRNAs. Microvesicles accumulated around immunoglobulin Fc receptor-positive (FcR+ ) cells. Upon internalisation, natural killer cells displayed significantly increased expression of genes related to activation and immune response, and downregulation of genes involved in the cell cycle. Cells may use microvesicles to transmit signals distally as part of a survival strategy. Microvesicles are equipped on their surface with enzymatic machinery leading to production of tolerogenic adenosine. Further, they are internalised in FcR+ cells with significant functional modifications. These observations have relevance for improving anti-CD38 therapeutic antibodies through targeting this mechanism and its sequelae.

Ectonucleotidase Expression on Human Amnion Epithelial Cells: Adenosinergic Pathways and Dichotomic Effects on Immune Effector Cell Populations.

This study investigates the mechanism(s) underlying the immunoregulatory activities of placenta-derived human amnion epithelial cells (hAEC). The working hypothesis is that NAD+ and ATP, along with ectoenzymes involved in their metabolism, play a significant role in hAEC-mediated immune regulation. Proof of principle of the hypothesis was obtained by analyzing the interactions between hAEC and the main human leukocyte populations. The results obtained indicate that hAEC constitutively express a unique combination of functional ectoenzymes, driving the production of adenosine (ADO) via canonical (CD39, CD73) and alternative (CD38, CD203a/PC-1, CD73) pathways. Further, the picture is completed by the observation that hAEC express A1, A2a, and A2b ADO receptors as well as ADO deaminase, the enzyme involved in ADO catabolism. The contribution of the purinergic mediator to immunomodulation was confirmed by exposing in vitro different immune effector cells to the action of primary hAECs. B cells showed an enhanced proliferation and diminished spontaneous apoptosis when in contact with hAEC. T cell proliferation was partially inhibited by hAEC through ADO production, as confirmed by using specific ectoenzyme inhibitors. Further, hAEC induced an expansion of both T and B regulatory cells. Last, hAEC inhibited NK cell proliferation. However, the involvement of ADO-producing ectoenzymes is less apparent in this context. In conclusion, hAEC exert different in vitro immunoregulatory effects, per se, as a result of interactions with different populations of immune effector cells. These results support the view that hAEC are instrumental for regenerative medicine as well as in therapeutic applications for immune-related diseases.

Dysfunctional Vγ9Vδ2 T cells are negative prognosticators and markers of dysregulated mevalonate pathway activity in chronic lymphocytic leukemia cells.

The role of Vγ9Vδ2 T cells in chronic lymphocytic leukemia (CLL) is unexplored, although these cells have a natural inclination to react against B-cell malignancies. Proliferation induced by zoledronic acid was used as a surrogate of γδ TCR-dependent stimulation to functionally interrogate Vγ9Vδ2 T cells in 106 untreated CLL patients. This assay permitted the identification of responder and low-responder (LR) patients. The LR status was associated with greater baseline counts of Vγ9Vδ2 T cells and to the expansion of the effector memory and terminally differentiated effector memory subsets. The tumor immunoglobulin heavy chain variable region was more frequently unmutated in CLL cells of LR patients, and the mevalonate pathway, which generates Vγ9Vδ2 TCR ligands, was more active in unmutated CLL cells. In addition, greater numbers of circulating regulatory T cells were detected in LR patients. In multivariate analysis, the LR condition was an independent predictor of shorter time-to-first treatment. Accordingly, the time-to-first treatment was significantly shorter in patients with greater baseline numbers of total Vγ9Vδ2 T cells and effector memory and terminally differentiated effector memory subpopulations. These results unveil a clinically relevant in vivo relationship between the mevalonate pathway activity of CLL cells and dys-functional Vγ9Vδ2 T cells.

The bone marrow of myeloma patients is steadily inhabited by a normal-sized pool of functional regulatory T cells irrespectiveof the disease status.

Conflicting data have been reported about the frequency and function of regulatory T cells in multiple myeloma. Most studies have investigated peripheral blood rather than bone marrow Tregs and side-by-side comparisons with bone marrow from healthy donors have still not been made. In this study, we show that regulatory T-cells total count, subset distribution, and expression of chemokine receptors are similar in the bone marrow of myeloma patients and healthy donors. Regulatory T cells are not recruited by myeloma cells in the bone marrow and their counts are unaffected by the tumor burden and the disease status. The diversity of T-cell receptor repertoire is highly preserved ensuring broad reactivity and effective suppressor function. Our results indicate that regulatory T cells may not be the main players of immunological tolerance to myeloma cells under base-line conditions, but their fully preserved immune competence may promote their inadvertent activation and blunt T-cell driven anti-myeloma immune interventions even after myeloma cells have successfully been cleared by chemotherapy.

Preclinical efficacy of carfilzomib in BRAF-mutant colorectal cancer models.

Serine/threonine-protein kinase B-raf (BRAF) mutations are found in 8-15% of colorectal cancer patients and identify a subset of tumors with poor outcome in the metastatic setting. We have previously reported that BRAF-mutant human cells display a high rate of protein production, causing proteotoxic stress, and are selectively sensitive to the proteasome inhibitors bortezomib and carfilzomib. In this work, we tested whether carfilzomib could restrain the growth of BRAF-mutant colorectal tumors not only by targeting cancer cells directly, but also by promoting an immune-mediated antitumor response. In human and mouse colorectal cancer cells, carfilzomib triggered robust endoplasmic reticulum stress and autophagy, followed by the emission of immunogenic-damage-associated molecules. Intravenous administration of carfilzomib delayed the growth of BRAF-mutant murine tumors and mobilized the danger-signal proteins calreticulin and high mobility group box 1 (HMGB1). Analyses of drug-treated samples revealed increased intratumor recruitment of activated cytotoxic T cells and natural killers, concomitant with the downregulation of forkhead box protein P3 (Foxp3)+ T-cell surface glycoprotein CD4 (CD4)+ T cells, indicating that carfilzomib promotes reshaping of the immune microenvironment of BRAF-mutant murine colorectal tumors. These results will inform the design of clinical trials in BRAF-mutant colorectal cancer patients.

Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition.

BACKGROUND: The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors. METHODS: We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells. RESULTS: MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells. CONCLUSIONS: Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

Immune modulation by zoledronic acid in human myeloma: an advantageous cross-talk between Vγ9Vδ2 T cells, αß CD8+ T cells, regulatory T cells, and dendritic cells.

Vγ9Vδ2 T cells play a major role as effector cells of innate immune responses against microbes, stressed cells, and tumor cells. They constitute <5% of PBLs but can be expanded by zoledronic acid (ZA)-treated monocytes or dendritic cells (DC). Much less is known about their ability to act as cellular adjuvants bridging innate and adaptive immunity, especially in patients with cancer. We have addressed this issue in multiple myeloma (MM), a prototypic disease with several immune dysfunctions that also affect γδ T cells and DC. ZA-treated MM DC were highly effective in activating autologous γδ T cells, even in patients refractory to stimulation with ZA-treated monocytes. ZA inhibited the mevalonate pathway of MM DC and induced the intracellular accumulation and release into the supernatant of isopentenyl pyrophosphate, a selective γδ T cell activator, in sufficient amounts to induce the proliferation of γδ T cells. Immune responses against the tumor-associated Ag survivin (SRV) by MHC-restricted, SRV-specific CD8(+) αß T cells were amplified by the concurrent activation of γδ T cells driven by autologous DC copulsed with ZA and SRV-derived peptides. Ancillary to the isopentenyl pyrophosphate-induced γδ T cell proliferation was the mevalonate-independent ZA ability to directly antagonize regulatory T cells and downregulate PD-L2 expression on the DC cell surface. In conclusion, ZA has multiple immune modulatory activities that allow MM DC to effectively handle the concurrent activation of γδ T cells and MHC-restricted CD8(+) αß antitumor effector T cells.

Exploring circular MET RNA as a potential biomarker in tumors exhibiting high MET activity.

BACKGROUND: MET-driven acquired resistance is emerging with unanticipated frequency in patients relapsing upon molecular therapy treatments. However, the determination of MET amplification remains challenging using both standard and next-generation sequencing-based methodologies. Liquid biopsy is an effective, non-invasive approach to define cancer genomic profiles, track tumor evolution over time, monitor treatment response and detect molecular resistance in advance. Circular RNAs (circRNAs), a family of RNA molecules that originate from a process of back-splicing, are attracting growing interest as potential novel biomarkers for their stability in body fluids. METHODS: We identified a circRNA encoded by the MET gene (circMET) and exploited blood-derived cell-free RNA (cfRNA) and matched tumor tissues to identify, stratify and monitor advanced cancer patients molecularly characterized by high MET activity, generally associated with genomic amplification. RESULTS: Using publicly available bioinformatic tools, we discovered that the MET locus transcribes several circRNA molecules, but only one candidate, circMET, was particularly abundant. Deeper molecular analysis revealed that circMET levels positively correlated with MET expression and activity, especially in MET-amplified cells. We developed a circMET-detection strategy and, in parallel, we performed standard FISH and IHC analyses in the same specimens to assess whether circMET quantification could identify patients displaying high MET activity. Longitudinal monitoring of circMET levels in the plasma of selected patients revealed the early emergence of MET amplification as a mechanism of acquired resistance to molecular therapies. CONCLUSIONS: We found that measurement of circMET levels allows identification and tracking of patients characterized by high MET activity. Circulating circMET (ccMET) detection and analysis could be a simple, cost-effective, non-invasive approach to better implement patient stratification based on MET expression, as well as to dynamically monitor over time both therapy response and clonal evolution during treatment.

Vγ9Vδ2 T cell-based immunotherapy in hematological malignancies: from bench to bedside.

Many hematological malignancies consist of tumor cells that are spontaneously recognized and killed by Vγ9Vδ2 T cells. These tumor cells generate high amounts of intracellular phosphorylated metabolites mimicking the natural ligands and display a wide range of stress-induced self-ligands that are recognized by Vγ9Vδ2 T cells via TCR-dependent and TCR-independent mechanisms. The intrinsic features of Vγ9Vδ2 T cells and that of tumor cells of hematological origin constitute an ideal combination from which to develop Vγ9Vδ2 T cell-based immune interventions. In this review, we will discuss the rationale, preclinical and clinical data in favor of this therapeutic strategy and the future perspectives of its development.

Immune dysfunctions affecting bone marrow Vγ9Vδ2 T cells in multiple myeloma: Role of immune checkpoints and disease status.

Introduction: Bone marrow (BM) Vγ9Vδ2 T cells are intrinsically predisposed to sense the immune fitness of the tumor microenvironment (TME) in multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Methods: In this work, we have used BM Vγ9Vδ2 T cells to interrogate the role of the immune checkpoint/immune checkpoint-ligand (ICP/ICP-L) network in the immune suppressive TME of MM patients. Results: PD-1+ BM MM Vγ9Vδ2 T cells combine phenotypic, functional, and TCR-associated alterations consistent with chronic exhaustion and immune senescence. When challenged by zoledronic acid (ZA) as a surrogate assay to interrogate the reactivity to their natural ligands, BM MM Vγ9Vδ2 T cells further up-regulate PD-1 and TIM-3 and worsen TCR-associated alterations. BM MM Vγ9Vδ2 T cells up-regulate TIM-3 after stimulation with ZA in combination with αPD-1, whereas PD-1 is not up-regulated after ZA stimulation with αTIM-3, indicating a hierarchical regulation of inducible ICP expression. Dual αPD-1/αTIM-3 blockade improves the immune functions of BM Vγ9Vδ2 T cells in MM at diagnosis (MM-dia), whereas single PD-1 blockade is sufficient to rescue BM Vγ9Vδ2 T cells in MM in remission (MM-rem). By contrast, ZA stimulation induces LAG-3 up-regulation in BM Vγ9Vδ2 T cells from MM in relapse (MM-rel) and dual PD-1/LAG-3 blockade is the most effective combination in this setting. Discussion: These data indicate that: 1) inappropriate immune interventions can exacerbate Vγ9Vδ2 T-cell dysfunction 2) ICP blockade should be tailored to the disease status to get the most of its beneficial effect.

Metabolic approaches to rescue antitumor Vγ9Vδ2 T-cell functions in myeloma.

Vγ9Vδ2 T cells are immune effector cells very well-suited for immunotherapy, but clinical results have been disappointing in multiple myeloma (MM) and other cancers. We have shown that Vg9Vd2 T cells are victimized prematurely by the immune suppressive tumor microenvironment (TME) established by myeloma and neighbouring cells in the bone marrow (BM) of MM patients. One major mechanism is the highly redundant expression of multiple immunecheckpoints/immune checkpoint-ligands (ICP/ICP-L) in the TME impairing antimyeloma Vg9Vd2 T-cell immune responses. Another major immune suppressive mechanism is the metabolic reset driven by myeloma cells in the TME to satisfy their energetic needs to the detriment of effector cells. Recently, it has become clear that the ICP/ICP-L circuitry and metabolic checkpoints (MCP) jointly operate in the TME of cancer patients to promote tumor cell growth and suppress antitumor immune responses. In this review, we discuss the possible interactions between ICP/ICP-L and MCP in the TME of MM patients that may compromise the immune competence of BM Vγ9Vδ2 T cells, envisaging novel combination therapies to improve the outcome of immune-based interventions.

ABCA1, apoA-I, and BTN3A1: A Legitimate Ménage à Trois in Dendritic Cells.

Human Vγ9Vδ2 T cells have the capacity to detect supra-physiological concentrations of phosphoantigens (pAgs) generated by the mevalonate (Mev) pathway of mammalian cells under specific circumstances. Isopentenyl pyrophosphate (IPP) is the prototypic pAg recognized by Vγ9Vδ2 T cells. B-cell derived tumor cells (i.e., lymphoma and myeloma cells) and dendritic cells (DCs) are privileged targets of Vγ9Vδ2 T cells because they generate significant amounts of IPP which can be boosted with zoledronic acid (ZA). ZA is the most potent aminobisphosphonate (NBP) clinically available to inhibit osteoclast activation and a very potent inhibitor of farnesyl pyrophosphate synthase in the Mev pathway. ZA-treated DCs generate and release in the supernatants picomolar IPP concentrations which are sufficient to induce the activation of Vγ9Vδ2 T cells. We have recently shown that the ATP-binding cassette transporter A1 (ABCA1) plays a major role in the extracellular release of IPP from ZA-treated DCs. This novel ABCA1 function is fine-tuned by physical interactions with IPP, apolipoprotein A-I (apoA-I), and butyrophilin-3A1 (BTN3A1). The mechanisms by which soluble IPP induces Vγ9Vδ2 T-cell activation remain to be elucidated. It is possible that soluble IPP binds to BTN3A1, apoA-I, or other unknown molecules on the cell surface of bystander cells like monocytes, NK cells, Vγ9Vδ2 T cells, or any other cell locally present. Investigating this scenario may represent a unique opportunity to further characterize the role of BTN3A1 and other molecules in the recognition of soluble IPP by Vγ9Vδ2 T cells.

Vγ9Vδ2 T Cells in the Bone Marrow of Myeloma Patients: A Paradigm of Microenvironment-Induced Immune Suppression.

Vγ9Vδ2 T cells are non-conventional T cells with a natural inclination to recognize and kill cancer cells. Malignant B cells, including myeloma cells, are privileged targets of Vγ9Vδ2 T cells in vitro. However, this inclination is often lost in vivo due to multiple mechanisms mediated by tumor cells and local microenvironment. Multiple myeloma (MM) is a paradigm disease in which antitumor immunity is selectively impaired at the tumor site. By interrogating the immune reactivity of bone marrow (BM) Vγ9Vδ2 T cells to phosphoantigens, we have revealed a very early and long-lasting impairment of Vγ9Vδ2 T-cell immune functions which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted even in clinical remission after autologous stem cell transplantation. Multiple cell subsets [MM cells, myeloid-derived suppressor cells, regulatory T cells, and BM-derived stromal cells (BMSC)] are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the redundant expression of multiple immune checkpoints (ICPs). This review will address some aspects related to the dynamics of ICP expression in the BM of MM patients in relationship to the disease status (MGUS, diagnosis, remission, and relapse) and how this multifaceted ICP expression impairs Vγ9Vδ2 T-cell function. We will also provide some suggestions how to rescue Vγ9Vδ2 T cells from the immune suppression operated by ICP and to recover their antimyeloma immune effector functions at the tumor site.

Vγ9Vδ2 T Cells as Strategic Weapons to Improve the Potency of Immune Checkpoint Blockade and Immune Interventions in Human Myeloma.

The advent of immune checkpoint (ICP) blockade has introduced an unprecedented paradigm shift in the treatment of cancer. Though very promising, there is still a substantial proportion of patients who do not respond or develop resistance to ICP blockade. In vitro and in vivo models are eagerly needed to identify mechanisms to maximize the immune potency of ICP blockade and overcome primary and acquired resistance to ICP blockade. Vγ9Vδ2 T cells isolated from the bone marrow (BM) from multiple myeloma (MM) are excellent tools to investigate the mechanisms of resistance to PD-1 blockade and to decipher the network of mutual interactions between PD-1 and the immune suppressive tumor microenvironment (TME). Vγ9Vδ2 T cells can easily be interrogated to dissect the progressive immune competence impairment generated in the TME by the long-lasting exposure to myeloma cellss. BM MM Vγ9Vδ2 T cells are PD-1+ and anergic to phosphoantigen (pAg) stimulation; notably, single agent PD-1 blockade is insufficient to fully recover their anti-tumor activity in vitro indicating that additional players are involved in the anergy of Vγ9Vδ2 T cells. In this mini-review we will discuss the value of Vγ9Vδ2 T cells as investigational tools to improve the potency of ICP blockade and immune interventions in MM.

Increasing intratumor C/EBP-ß LIP and nitric oxide levels overcome resistance to doxorubicin in triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) easily develops resistance to the first-line drug doxorubicin, because of the high levels of the drug efflux transporter P-glycoprotein (Pgp) and the activation of pro-survival pathways dependent on endoplasmic reticulum (ER). Interfering with these mechanisms may overcome the resistance to doxorubicin, a still unmet need in TNBC. METHODS: We analyzed a panel of human and murine breast cancer cells for their resistance to doxorubicin, Pgp expression, lysosome and proteasome activity, nitrite production, ER-dependent cell death and immunogenic cell death parameters. We evaluated the efficacy of genetic (C/EBP-ß LIP induction) and pharmacological strategies (lysosome and proteasome inhibitors), in restoring the ER-dependent and immunogenic-dependent cell death induced by doxorubicin, in vitro and in syngeneic mice bearing chemoresistant TNBC. The results were analyzed by one-way analysis of variance test. RESULTS: We found that TNBC cells characterized by high levels of Pgp and resistance to doxorubicin, had low induction of the ER-dependent pro-apoptotic factor C/EBP-ß LIP upon doxorubicin treatment and high activities of lysosome and proteasome that constitutively destroyed LIP. The combination of chloroquine and bortezomib restored doxorubicin sensitivity by activating multiple and interconnected mechanisms. First, chloroquine and bortezomib prevented C/EBP-ß LIP degradation and activated LIP-dependent CHOP/TRB3/caspase 3 axis in response to doxorubicin. Second, C/EBP-ß LIP down-regulated Pgp and up-regulated calreticulin that triggered the dendritic cell (DC)-mediated phagocytosis of tumor cell, followed by the activation of anti-tumor CD8+T-lymphocytes upon doxorubicin treatment. Third, chloroquine and bortezomib increased the endogenous production of nitric oxide that further induced C/EBP-ß LIP and inhibited Pgp activity, enhancing doxorubicin's cytotoxicity. In orthotopic models of resistant TNBC, intratumor C/EBP-ß LIP induction - achieved by a specific expression vector or by chloroquine and bortezomib - effectively reduced tumor growth and Pgp expression, increased intra-tumor apoptosis and anti-tumor immune-infiltrate, rescuing the efficacy of doxorubicin. CONCLUSIONS: We suggest that preventing C/EBP-ß LIP degradation by lysosome and proteasome inhibitors triggers multiple virtuous circuitries that restore ER-dependent apoptosis, down-regulate Pgp and re-activate the DC/CD8+T-lymphocytes response against TNBC. Lysosome and proteasome inhibitors associated with doxorubicin may overcome the resistance to the drug in TNBC.

The ATP-binding cassette transporter A1 regulates phosphoantigen release and Vγ9Vδ2 T cell activation by dendritic cells.

Vγ9Vδ2 T cells are activated by phosphoantigens, such as isopentenyl pyrophosphate (IPP), which is generated in the mevalonate pathway of antigen-presenting cells. IPP is released in the extracellular microenvironment via unknown mechanisms. Here we show that the ATP-binding cassette transporter A1 (ABCA1) mediates extracellular IPP release from dendritic cells (DC) in cooperation with apolipoprotein A-I (apoA-I) and butyrophilin-3A1. IPP concentrations in the supernatants are sufficient to induce Vγ9Vδ2 T cell proliferation after DC mevalonate pathway inhibition with zoledronic acid (ZA). ZA treatment increases ABCA1 and apoA-I expression via IPP-dependent LXRα nuclear translocation and PI3K/Akt/mTOR pathway inhibition. These results close the mechanistic gap in our understanding of extracellular IPP release from DC and provide a framework to fine-tune Vγ9Vδ2 T cell activation via mevalonate and PI3K/Akt/mTOR pathway modulation.