The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition.

Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment. To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin. These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.

p140Cap modulates the mevalonate pathway decreasing cell migration and enhancing drug sensitivity in breast cancer cells.

p140Cap is an adaptor protein involved in assembling multi-protein complexes regulating several cellular processes. p140Cap acts as a tumor suppressor in breast cancer (BC) and neuroblastoma patients, where its expression correlates with a better prognosis. The role of p140Cap in tumor metabolism remains largely unknown. Here we study the role of p140Cap in the modulation of the mevalonate (MVA) pathway in BC cells. The MVA pathway is responsible for the biosynthesis of cholesterol and non-sterol isoprenoids and is often deregulated in cancer. We found that both in vitro and in vivo, p140Cap cells and tumors show an increased flux through the MVA pathway by positively regulating the pace-maker enzyme of the MVA pathway, the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR), via transcriptional and post-translational mechanisms. The higher cholesterol synthesis is paralleled with enhanced cholesterol efflux. Moreover, p140Cap promotes increased cholesterol localization in the plasma membrane and reduces lipid rafts-associated Rac1 signalling, impairing cell membrane fluidity and cell migration in a cholesterol-dependent manner. Finally, p140Cap BC cells exhibit decreased cell viability upon treatments with statins, alone or in combination with chemotherapeutic at low concentrations in a synergistic manner. Overall, our data highlight a new perspective point on tumor suppression in BC by establishing a previously uncharacterized role of the MVA pathway in p140Cap expressing tumors, thus paving the way to the use of p140Cap as a potent biomarker to stratify patients for better tuning therapeutic options.

4-(3-Phenyl-4-(3,4,5-trimethoxybenzoyl)-1H-pyrrol-1-yl)benzenesulfonamide, a Novel Carbonic Anhydrase and Wnt/ß-Catenin Signaling Pathway Dual-Targeting Inhibitor with Potent Activity against Multidrug Resistant Cancer Cells.

We synthesized new pyrrole and indole derivatives as human carbonic anhydrase (hCA) inhibitors with the potential to inhibit the Wnt/ß-catenin signaling pathway. The presence of both N1-(4-sulfonamidophenyl) and 3-(3,4,5-trimethoxyphenyl) substituents was essential for strong hCA inhibitors. The most potent hCA XII inhibitor 15 (Ki = 6.8 nM) suppressed the Wnt/ß-catenin signaling pathway and its target genes MYC, Fgf20, and Sall4 and exhibited the typical markers of apoptosis, cleaved poly(ADP-ribose)polymerase, and cleaved caspase-3. Compound 15 showed strong inhibition of viability in a panel of cancer cells, including colorectal cancer and triple-negative breast cancer cells, was effective against the NCI/ADR-RES DOX-resistant cell line, and restored the sensitivity to doxorubicin (DOX) in HT29/DX and MDCK/P-gp cells. Compound 15 is a novel dual-targeting compound with activity against hCA and Wnt/ß-catenin. It thus has a broad targeting spectrum and is an anticancer agent with specific potential in P-glycoprotein overexpressing cell lines.

Cytotoxic pathways activated by multifunctional thiosemicarbazones targeting sigma-2 receptors in breast and lung carcinoma cells.

BACKGROUND: Multifunctional thiosemicarbazones (TSCs) able to bind sigma receptors and chelate metals are considered as a promising avenue for the treatment of pancreatic cancer due to the encouraging results obtained on in vitro and in vivo models. Here, we assessed the biochemical mechanism of these TSCs also on lung (A549) and breast (MCF7) cancer cells. METHODS: The density of sigma-2 receptors in normal (BEAS-2B and MCF10A) and in lung and breast (A549 and MCF7) cancer cells was evaluated by flow cytometry. In these cells, cytotoxicity (MTT assay) and activation of ER- and mitochondria-dependent cell death pathways (by spectrofluorimetric assays to measure Caspases 3/7/9; qRT-PCR detection of GRP78, ATF6, IRE1, PERK; MitoSOX, DCFDA-AM and JC-1 staining), induced by the TSCs FA4, MLP44, PS3 and ACThio1, were evaluated. RESULTS: FA4 and PS3 exerted more potent cytotoxicity than MLP44 and ACThio1 in all cancer cell lines, where the density of sigma-2 receptors was higher than in normal cells. Remarkably, FA4 promoted ER- and mitochondria-dependent cell death pathways in both cell models, whereas the other TSCs had variable, cell-dependent effects on the activation of the two proapoptotic pathways. CONCLUSIONS: Our data suggest that FA4 is a promising compound that deserves to be further studied for lung and breast cancer treatment. However, the other multifunctional TSCs also hold promise for the development of therapies towards a personalized medicine approach. Indeed, the presence of the sigma-2 receptor-targeting moiety would lead to a more specific tumor delivery embracing the characteristics of individual tumor types.

Autocrine 17-ß-Estradiol/Estrogen Receptor-α Loop Determines the Response to Immune Checkpoint Inhibitors in Non-Small Cell Lung Cancer.

PURPOSE: The response to immune checkpoint inhibitors (ICI) often differs between genders in non-small cell lung cancer (NSCLC), but metanalyses results are controversial, and no clear mechanisms are defined. We aim at clarifying the molecular circuitries explaining the differential gender-related response to anti-PD-1/anti-PD-L1 agents in NSCLC. EXPERIMENTAL DESIGN: We prospectively analyzed a cohort of patients with NSCLC treated with ICI as a first-line approach, and we identified the molecular mechanisms determining the differential efficacy of ICI in 29 NSCLC cell lines of both genders, recapitulating patients' phenotype. We validated new immunotherapy strategies in mice bearing NSCLC patient-derived xenografts and human reconstituted immune system ("immune-PDXs"). RESULTS: In patients, we found that estrogen receptor α (ERα) was a predictive factor of response to pembrolizumab, stronger than gender and PD-L1 levels, and was directly correlated with PD-L1 expression, particularly in female patients. ERα transcriptionally upregulated CD274/PD-L1 gene, more in females than in males. This axis was activated by 17-ß-estradiol, autocrinely produced by intratumor aromatase, and by the EGFR-downstream effectors Akt and ERK1/2 that activated ERα. The efficacy of pembrolizumab in immune-PDXs was significantly improved by the aromatase inhibitor letrozole, which reduced PD-L1 and increased the percentage of antitumor CD8+T-lymphocytes, NK cells, and Vγ9Vδ2 T-lymphocytes, producing durable control and even tumor regression after continuous administration, with maximal benefit in 17-ß-estradiol/ERα highfemale immune-xenografts. CONCLUSIONS: Our work unveils that 17-ß-estradiol/ERα status predicts the response to pembrolizumab in patients with NSCLC. Second, we propose aromatase inhibitors as new gender-tailored immune-adjuvants in NSCLC. See related commentary by Valencia et al., p. 3832.

Long-Chain Acyl Coenzyme A Dehydrogenase, a Key Player in Metabolic Rewiring/Invasiveness in Experimental Tumors and Human Mesothelioma Cell Lines.

Cross-species investigations of cancer invasiveness are a new approach that has already identified new biomarkers which are potentially useful for improving tumor diagnosis and prognosis in clinical medicine and veterinary science. In this study, we combined proteomic analysis of four experimental rat malignant mesothelioma (MM) tumors with analysis of ten patient-derived cell lines to identify common features associated with mitochondrial proteome rewiring. A comparison of significant abundance changes between invasive and non-invasive rat tumors gave a list of 433 proteins, including 26 proteins reported to be exclusively located in mitochondria. Next, we analyzed the differential expression of genes encoding the mitochondrial proteins of interest in five primary epithelioid and five primary sarcomatoid human MM cell lines; the most impressive increase was observed in the expression of the long-chain acyl coenzyme A dehydrogenase (ACADL). To evaluate the role of this enzyme in migration/invasiveness, two epithelioid and two sarcomatoid human MM cell lines derived from patients with the highest and lowest overall survival were studied. Interestingly, sarcomatoid vs. epithelioid cell lines were characterized by higher migration and fatty oxidation rates, in agreement with ACADL findings. These results suggest that evaluating mitochondrial proteins in MM specimens might identify tumors with higher invasiveness. Data are available via ProteomeXchange with the dataset identifier PXD042942.

The role of Extracellular Vesicles in glycolytic and lipid metabolic reprogramming of cancer cells: Consequences for drug resistance.

In order to adapt to a higher proliferative rate and an increased demand for energy sources, cancer cells rewire their metabolic pathways, a process currently recognized as a hallmark of cancer. Even though the metabolism of glucose is perhaps the most discussed metabolic shift in cancer, lipid metabolic alterations have been recently recognized as relevant players in the growth and proliferation of cancer cells. Importantly, some of these metabolic alterations are reported to induce a drug resistant phenotype in cancer cells. The acquisition of drug resistance traits severely hinders cancer treatment, being currently considered one of the major challenges of the oncological field. Evidence suggests that Extracellular Vesicles (EVs), which play a crucial role in intercellular communication, may act as facilitators of tumour progression, survival and drug resistance by modulating several aspects involved in the metabolism of cancer cells. This review aims to gather and discuss relevant data regarding metabolic reprograming in cancer, particularly involving the glycolytic and lipid alterations, focusing on its influence on drug resistance and highlighting the relevance of EVs as intercellular mediators of this process.

The multiple combination of Paclitaxel, Ramucirumab and Elacridar reverses the paclitaxel-mediated resistance in gastric cancer cell lines.

Introduction: Paclitaxel (PTX) interferes with microtubule architecture by binding to ß-tubulin, thereby blocking progression at the G2/M phase and inducing apoptosis. This study aimed to investigate molecular processes underlying PTX-mediated resistance in gastric cancer (GC) cells. Methods: PTX-mediated resistance involves many processes, and in this work some of the factors involved in the resistance mechanism were identified by comparing two GC lines with PTX induced resistance to their sensitive counterparts. Results: Thus, the key feature of PTX-resistant cells was the overexpression of pro-angiogenic factors such as VEGFA, VEGFC, and Ang2, known to support tumor cell growth. A second relevant change detected in PTX-resistant lines was the elevated level of TUBßIII, a tubulin isoform that opposes microtubule stabilization. A third identified factor contributing to PTX-resistance was P-glycoprotein (P-gp), a transporter responsible for chemotherapy efflux from the cells, highly expressed in PTX-resistant lines. Discussion: These findings were in line with a greater sensitivity of resistant cells to treatment with both Ramucirumab and Elacridar. Ramucirumab significantly reduced the expression of angiogenic molecules and TUBßIII, while Elacridar restored the access of chemotherapy, recovering its anti-mitotic and pro-apoptotic effects. Finally, this study highlighted the role played by exosomes in spreading factors responsible for resistance in the tumor microenvironment.

Strategies to Overcome Resistance to Immune-Based Therapies in Osteosarcoma.

Improving the prognosis and cure rate of HGOSs (high-grade osteosarcomas) is an absolute need. Immune-based treatment approaches have been increasingly taken into consideration, in particular for metastatic, relapsed and refractory HGOS patients, to ameliorate the clinical results currently achieved. This review is intended to give an overview on the immunotherapeutic treatments targeting, counteracting or exploiting the different immune cell compartments that are present in the HGOS tumor microenvironment. The principle at the basis of these strategies and the possible mechanisms that HGOS cells may use to escape these treatments are presented and discussed. Finally, a list of the currently ongoing immune-based trials in HGOS is provided, together with the results that have been obtained in recently completed clinical studies. The different strategies that are presently under investigation, which are generally aimed at abrogating the immune evasion of HGOS cells, will hopefully help to indicate new treatment protocols, leading to an improvement in the prognosis of patients with this tumor.

mTORC2 Is Activated under Hypoxia and Could Support Chronic Myeloid Leukemia Stem Cells.

Hypoxia is a critical condition that governs survival, self-renewal, quiescence, metabolic shift and refractoriness to leukemic stem cell (LSC) therapy. The present study aims to investigate the hypoxia-driven regulation of the mammalian Target of the Rapamycin-2 (mTORC2) complex to unravel it as a novel potential target in chronic myeloid leukemia (CML) therapeutic strategies. After inducing hypoxia in a CML cell line model, we investigated the activities of mTORC1 and mTORC2. Surprisingly, we detected a significant activation of mTORC2 at the expense of mTORC1, accompanied by the nuclear localization of the main substrate phospho-Akt (Ser473). Moreover, the Gene Ontology analysis of CML patients' CD34+ cells showed enrichment in the mTORC2 signature, further strengthening our data. The deregulation of mTOR complexes highlights how hypoxia could be crucial in CML development. In conclusion, we propose a mechanism by which CML cells residing under a low-oxygen tension, i.e., in the leukemia quiescent LSCs, singularly regulate the mTORC2 and its downstream effectors.

Mitochondrial ROS drive resistance to chemotherapy and immune-killing in hypoxic non-small cell lung cancer.

BACKGROUND: Solid tumors subjected to intermittent hypoxia are characterized by resistance to chemotherapy and immune-killing by effector T-lymphocytes, particularly tumor-infiltrating Vγ9Vδ2 T-lymphocytes. The molecular circuitries determining this double resistance are not known. METHODS: We analyzed a panel of 28 human non-small cell lung cancer (NSCLC) lines, using an in vitro system simulating continuous and intermittent hypoxia. Chemosensitivity to cisplatin and docetaxel was evaluated by chemiluminescence, ex vivo Vγ9Vδ2 T-lymphocyte expansion and immune-killing by flow cytometry. Targeted transcriptomics identified efflux transporters and nuclear factors involved in this chemo-immuno-resistance. The molecular mechanism linking Hypoxia-inducible factor-1α (HIF-1α), CCAAT/Enhancer Binding Protein-ß (C/EBP-ß) isoforms LAP and LIP, ABCB1, ABCC1 and ABCA1 transporters were evaluated by immunoblotting, RT-PCR, RNA-IP, ChIP. Oxidative phosphorylation, mitochondrial ATP, ROS, depolarization, O2 consumption were monitored by spectrophotometer and electronic sensors. The role of ROS/HIF-1α/LAP axis was validated in knocked-out or overexpressing cells, and in humanized (Hu-CD34+NSG) mice bearing LAP-overexpressing tumors. The clinical meaning of LAP was assessed in 60 NSCLC patients prospectively enrolled, treated with chemotherapy. RESULTS: By up-regulating ABCB1 and ABCC1, and down-regulating ABCA1, intermittent hypoxia induced a stronger chemo-immuno-resistance than continuous hypoxia in NSCLC cells. Intermittent hypoxia impaired the electron transport chain and reduced O2 consumption, increasing mitochondrial ROS that favor the stabilization of C/EBP-ß mRNA mediated by HIF-1α. HIF-1α/C/EBP-ß mRNA binding increases the splicing of C/EBP-ß toward the production of LAP isoform that transcriptionally induces ABCB1 and ABCC1, promoting the efflux of cisplatin and docetaxel. LAP also decreases ABCA1, limiting the efflux of isopentenyl pyrophosphate, i.e. the endogenous activator of Vγ9Vδ2 T-cells, and reducing the immune-killing. In NSCLC patients subjected to cisplatin-based chemotherapy, C/EBP-ß LAP was abundant in hypoxic tumors and was associated with lower response to treatment and survival. LAP-overexpressing tumors in Hu-CD34+NSG mice recapitulated the patients' chemo-immuno-resistant phenotype. Interestingly, the ROS scavenger mitoquinol chemo-immuno-sensitized immuno-xenografts, by disrupting the ROS/HIF-1α/LAP cascade. CONCLUSIONS: The impairment of mitochondrial metabolism induced by intermittent hypoxia increases the ROS-dependent stabilization of HIF-1α/LAP complex in NSCLC, producing chemo-immuno-resistance. Clinically used mitochondrial ROS scavengers may counteract such double resistance. Moreover, we suggest C/EBP-ß LAP as a new predictive and prognostic factor in NSCLC patients.

Electronic Coupling in 1,2,3-Triazole Bridged Ferrocenes and Its Impact on Reactive Oxygen Species Generation and Deleterious Activity in Cancer Cells.

Mixed-valence (MV) binuclear ferrocenyl compounds have long been studied as models for testing theories of electron transfer and in attempts to design molecular-scale electronic devices (e.g., molecular wires). In contrary to that, far less attention has been paid to MV binuclear ferrocenes as anticancer agents. Herein, we discuss the synthesis of six 1,2,3-triazole ferrocenyl compounds for combined (spectro)electrochemical, electron paramagnetic resonance (EPR), computational, and anticancer activity studies. Our synthetic approach was based on the copper-catalyzed 1,3-dipolar azide-alkyne cycloaddition reaction and enabled us to obtain in one step compounds bearing either one, two, or three ferrocenyl entities linked to the common 1,2,3-triazole core. Thus, two series of complexes were obtained, which pertain to derivatives of 3'-azido-3'-deoxythymidine (AZT) and 3-azidopropionylferrocene, respectively. Based on the experimental and theoretical data, the two mono-oxidized species corresponding to binuclear AZT and trinuclear 3-azidopropionylferrocene complexes have been categorized as class II mixed-valence according to the classification proposed by Robin and Day. Of importance is the observation that these two compounds are more active against human A549 and H1975 non-small-cell lung cancer cells than their congeners, which do not show MV characteristics. Moreover, the anticancer activity of MV species competes or surpasses, dependent on the cell line, the activity of reference anticancer drugs such as cisplatin, tamoxifen, and 5-fluorouracil. The most active from the entire series of compounds was the binuclear thymidine derivative with the lowest IC50 value of 5 ± 2 µM against lung H1975 cancer cells. The major mechanism of antiproliferative activity for the investigated MV compounds is based on reactive oxygen species generation in cancer cells. This hypothesis was substantiated by EPR spin-trapping experiments and the observation of decreased anticancer activity in the presence of N-acetyl cysteine (NAC) free-radical scavenger.

The role of extracellular vesicles in the transfer of drug resistance competences to cancer cells.

Drug resistance remains a major hurdle to successful cancer treatment, being accountable for approximately 90% of cancer-related deaths. In the past years, increasing attention has been given to the role of extracellular vesicles (EVs) in the horizontal transfer of drug resistance in cancer. Indeed, many studies have described the dissemination of therapy resistance traits mediated by EVs, which may be transferred from drug resistant tumor cells to their drug sensitive counterparts. Importantly, different key players of drug resistance have been identified in the cargo of those EVs, such as drug efflux pumps, oncoproteins, antiapoptotic proteins, or microRNAs, among others. Interestingly, the EVs-mediated crosstalk between cells from the tumor microenvironment (TME) and tumor cells has emerged as another important mechanism that leads to cancer cells drug resistance. Recently, the cargo of the TME-derived EVs responsible for the transfer of drug resistance traits has also become a focus of attention. In addition, the possible mechanisms involved in drug sequestration by EVs, likely to contribute to cancer drug resistance, are also described and discussed herein. Despite the latest scientific advances in the field of EVs, this is still a challenging area of research, particularly in the clinical setting. Therefore, further investigation is needed to assess the relevance of EVs to the failure of cancer patients to drug treatment, to identify biomarkers of drug resistance in the EV's cargo, and to develop effective therapeutic strategies to surmount drug resistance. This up-to-date review summarizes relevant literature on the role of EVs in the transfer of drug resistance competences to cancer cells, and the relevance of tumor cells and of TME cells in this process. Finally, this knowledge is integrated with a discussion of possible future clinical applications of EVs as biomarkers of drug resistance.

SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma.

BACKGROUND: The combination of pemetrexed and cisplatin remains the reference first-line systemic therapy for malignant pleural mesothelioma (MPM). Its activity is moderate because of tumor aggressiveness, immune-suppressive environment and resistance to chemotherapy-induced immunogenic cell death (ICD). Preliminary and limited findings suggest that MPM cells have deregulated ubiquitination and proteasome activities, although proteasome inhibitors achieved disappointing clinical results. METHODS: Here, we investigated the role of the E3-ubiquitin ligase SKP/Cullin/F-box (SCF) complex in cell cycle progression, endoplasmic reticulum (ER)/proteostatic stress and ICD in MPM, and the therapeutic potential of the neddylation/SCF complex inhibitor MLN4924/Pevonedistat. RESULTS: In patient-derived MPM cultures and syngenic murine models, MLN4924 and cisplatin showed anti-tumor effects, regardless of MPM histotype and BAP1 mutational status, increasing DNA damage, inducing S- and G2/M-cell cycle arrest, and apoptosis. Mechanistically, by interfering with the neddylation of cullin-1 and ubiquitin-conjugating enzyme UBE2M, MLN4924 blocks the SCF complex activity and triggers an ER stress-dependent ICD, which activated anti-MPM CD8+T-lymphocytes. The SKP2 component of SCF complex was identified as the main driver of sensitivity to MLN4924 and resistance to cisplatin. These findings were confirmed in a retrospective MPM patient series, where SKP2 high levels were associated with a worse response to platinum-based therapy and inferior survival. CONCLUSIONS: We suggest that the combination of neddylation inhibitors and cisplatin could be worth of further investigation in the clinical setting for MPM unresponsive to cisplatin. We also propose SKP2 as a new stratification marker to determine the sensitivity to cisplatin and drugs interfering with ubiquitination/proteasome systems in MPM.

Targeted Self-Emulsifying Drug Delivery Systems to Restore Docetaxel Sensitivity in Resistant Tumors.

The use of chemotherapeutic agents such as docetaxel (DTX) in anticancer therapy is often correlated to side effects and the occurrence of drug resistance, which substantially impair the efficacy of the drug. Here, we demonstrate that self-emulsifying drug delivery systems (SEDDS) coated with enoxaparin (Enox) are a promising strategy to deliver DTX in resistant tumors. DTX partition studies between the SEDDS pre-concentrate and the release medium (water) suggest that the drug is well retained within the SEDDS upon dilution in the release medium. All SEDDS formulations show droplets with a mean diameter between 110 and 145 nm following dilution in saline and negligible hemolytic activity; the droplet size remains unchanged upon sterilization. Enox-coated SEDDS containing DTX exhibit an enhanced inhibition of cell growth compared to the control on cells of different solid tumors characterized by high levels of FGFR, which is due to an increased DTX internalization mediated by Enox. Moreover, only Enox-coated SEDDS are able to restore the sensitivity to DTX in resistant cells expressing MRP1 and BCRP by inhibiting the activity of these two main efflux transporters for DTX. The efficacy and safety of these formulations is also confirmed in vivo in resistant non-small cell lung cancer xenografts.

Mitochondria-Targeted Drug Delivery.

Mitochondria, organelles surrounded by a double membrane and with their own small genome, are the cells' energy centres [...].

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.

Glabratephrin reverses doxorubicin resistance in triple negative breast cancer by inhibiting P-glycoprotein.

Triple-negative breast cancer is one of the most aggressive breast cancer. The first therapeutic option is chemotherapy, often based on anthracycline as doxorubicin. However, chemotherapy efficacy is limited in by the presence of P-glycoprotein (Pgp), a membrane transporter protein that effluxes doxorubicin, reducing its cellular accumulation and toxicity. Inhibiting Pgp activity with effective and non-toxic products is still an open challenge. In this work, we demonstrated that the natural product Glabratephrin (Glab), a prenylated flavonoid from Tephrosia purpurea with a unique chemical structure, increased doxorubicin accumulation and cytotoxicity in triple negative breast cancer cells with high levels of Pgp, characterized by both acquired or intrinsic resistance to doxorubicin. Glab also reduced the growth of Pgp-expressing tumors, without adding significant extra-toxicities to doxorubicin treatment. Interestingly, Glab did not change the expression of Pgp, but it reduced the affinity for Pgp and the efflux of doxorubicin, as suggested by the increased Km and the reduced Vmax. In silico molecular docking predicted that Glab binds two residues (phenylalanine 322, glutamine 721) localized in the transmembrane domains of Pgp, facing the extracellular environment. Moreover, site-directed mutagenesis identified glycine 185 as a critical residue mediating the reduced catalytic efficacy of Pgp elicited by Glab. We propose Glab as an effective and safe compound able to reverse doxorubicin resistance mediated by Pgp in triple negative breast cancers, opening the way to a new combinatorial approach that may improve chemotherapy efficacy in the most refractory and aggressive breast cancer.