Metronomic cyclophosphamide attenuates mTOR-mediated expansion of regulatory T cells, but does not impact clinical outcome in patients with metastatic renal cell cancer treated with everolimus.

INTRODUCTION: Metastatic renal cell cancer (mRCC) patients have a median overall survival (mOS) of approximately 28 months. Until recently, mammalian target of rapamycin (mTOR) inhibition with everolimus was the standard second-line treatment regimen for mRCC patients, improving median progression-free survival (mPFS). Treatment with everolimus supports the expansion of immunosuppressive regulatory T cells (Tregs), which exert a negative effect on antitumor immune responses. In a phase 1 dose-escalation study, we have recently demonstrated that a low dose of 50 mg oral cyclophosphamide once daily can be safely combined with everolimus in mRCC patients and prevents the everolimus-induced increase in Tregs. MATERIALS AND METHODS: In a multicenter phase 2 study, performed in patients with mRCC not amenable to or progressive on a vascular endothelial growth factor (VEGF)-receptor tyrosine kinase inhibitor (TKI) containing treatment regimen, we assessed whether the addition of this metronomic dosing schedule of cyclophosphamide to therapy with everolimus could result in an improvement of progression-free survival (PFS) after 4 months of treatment. RESULTS: Though results from this study confirmed that combination treatment effectively lowered circulating levels of Tregs, addition of cyclophosphamide did not improve the PFS rate at 4 months. For this reason, the study was abrogated at the predefined interim analysis. CONCLUSION: Although the comprehensive immunomonitoring analysis performed in this study provides relevant information for the design of future immunotherapeutic approaches, the addition of metronomic cyclophosphamide to mRCC patients receiving everolimus cannot be recommended.

The effect of everolimus and low-dose cyclophosphamide on immune cell subsets in patients with metastatic renal cell carcinoma: results from a phase I clinical trial.

For the treatment of metastatic renal cell cancer several strategies are used among which the mTOR inhibitor everolimus. As mTOR plays an important role in the immune system, e.g., by controlling the expression of the transcription factor FoxP3 thereby regulating regulatory T cells (Tregs), it plays a key role in the balance between tolerance and inflammation. Previous reports showed stimulatory effects of mTOR inhibition on the expansion of Tregs, an effect that can be considered detrimental in terms of cancer control. Since metronomic cyclophosphamide (CTX) was shown to selectively deplete Tregs, a phase 1 clinical trial was conducted to comprehensively investigate the immune-modulating effects of several dosages and schedules of CTX in combination with the standard dose of everolimus, with the explicit aim to achieve selective Treg depletion. Our data show that 50 mg of CTX once daily and continuously administered, in combination with the standard dose of 10 mg everolimus once daily, not only results in depletion of Tregs, but also leads to a reduction in MDSC, a sustained level of the CD8+ T-cell population accompanied by an increased effector to suppressor ratio, and reversal of negative effects on three peripheral blood DC subsets. These positive effects on the immune response may contribute to improved survival, and therefore this combination therapy is further evaluated in a phase II clinical trial.

Phase 1 study of everolimus and low-dose oral cyclophosphamide in patients with metastatic renal cell carcinoma.

mTOR inhibitors are frequently used in the treatment of metastatic renal cell cancer (mRCC). mTOR regulates cell growth, proliferation, angiogenesis, and survival, and additionally plays an important role in immune regulation. Since mTOR inhibitors were shown to benefit immunosuppressive regulatory T-cell (Treg) expansion, this might suppress antitumor immune responses. Metronomic cyclophosphamide (CTX) was shown to selectively deplete Tregs. This study was, therefore, designed to determine the optimal dosage and schedule of CTX when combined with everolimus to prevent this potentially detrimental Treg expansion. In this national multi-center phase I study, patients with mRCC progressive on first line anti-angiogenic therapy received 10 mg everolimus once daily and were enrolled into cohorts with different CTX dosages and schedules. Besides immune monitoring, adverse events and survival data were monitored. 40 patients, 39 evaluable, were treated with different doses and schedules of CTX. Combined with 10 mg everolimus once daily, the optimal Treg depleting dose and schedule of CTX was 50 mg CTX once daily. 23 (59%) patients experienced one or more treatment-related ≥ grade 3 toxicity, mostly fatigue, laboratory abnormalities and pneumonitis. The majority of the patients achieved stable disease, two patients a partial response. Median PFS of all cohorts was 3.5 months. In conclusion, the optimal Treg depleting dose and schedule of CTX, when combined with everolimus, is 50 mg once daily. This combination leads to acceptable adverse events in comparison with everolimus alone. Currently, the here selected combination is being evaluated in a phase II clinical trial. TRIAL REGISTRATION: NCT01462214.

The effects of systemic treatment with aminobisphosphonates and statins on circulating Vγ9Vδ2-T cells in patients with advanced cancer.

Aminobisphosphonates (NBP) are used for treatment of metastatic bone disease. Frequently, patients undergoing NBP-treatment experience side-effects, known as acute phase response (APR), resulting from cytokine production by Vγ9Vδ2-T cells. As opposed to NBP, statins reduce intracellular phosphoantigen levels and prevent NBP-induced Vγ9Vδ2-T cell activation in vitro. We conducted a pilot study in patients with (bone-)metastasized malignancies receiving NBP-treatment and evaluated the phenotype and function of circulating Vγ9Vδ2-T cells in vivo and the effects of statins on Vγ9Vδ2-T cell responses and the associated APR. We observed reduced expression of perforin, granzyme B and HLA-DR on Vγ9Vδ2-T cells in patients treated with NBP and statins. However, statins could not prevent NBP-induced changes in circulating Vγ9Vδ2-T cell numbers or production of IFNγ and TNFα. Consistent with this, simvastatin could not prevent the occurrence of APR upon NBP-infusion. These observations call for the exploration of alternative strategies to prevent collateral APR upon NBP treatment.

Immunological effects of everolimus in patients with metastatic renal cell cancer.

The mammalian target of rapamycin (mTOR) is a crucial kinase present in all cells. Besides its role in the regulation of cell-growth, proliferation, angiogenesis, and survival of malignant tumors, mTOR additionally plays an important role in immune regulation by controlling the balance between effector T cells and regulatory T cells (Tregs). This critically affects the suppressive state of the immune system. Here, the systemic immunological effects of everolimus treatment were comprehensively investigated in five patients with metastatic renal cell cancer. In this hypothesis generating study, the immunological alterations in circulating immune subsets induced by everolimus included a (non-significant) increase in the frequency of Tregs, a significant increase in monocytic myeloid-derived suppressor cells, a significant decrease in the frequency of immunoregulatory natural killer cells, classical CD141+ (cDC1) and CD1c+ (cDC2) dendritic cell subsets, as well as a decrease in the activation status of plasmacytoid dendritic cells and cDC1. These date indicate that the immunological effects of everolimus affect multiple immune cell subsets and altogether tip the balance in favor of immunosuppression, which can be considered a detrimental effect in the treatment of cancer, and may require combination treatment with agents able to negate immune suppression and boost T cell immunity.

Evaluation of a tyrosine kinase peptide microarray for tyrosine kinase inhibitor therapy selection in cancer.

Personalized cancer medicine aims to accurately predict the response of individual patients to targeted therapies, including tyrosine kinase inhibitors (TKIs). Clinical implementation of this concept requires a robust selection tool. Here, using both cancer cell lines and tumor tissue from patients, we evaluated a high-throughput tyrosine kinase peptide substrate array to determine its readiness as a selection tool for TKI therapy. We found linearly increasing phosphorylation signal intensities of peptides representing kinase activity along the kinetic curve of the assay with 7.5-10 µg of lysate protein and up to 400 µM adenosine triphosphate (ATP). Basal kinase activity profiles were reproducible with intra- and inter-experiment coefficients of variation of <15% and <20%, respectively. Evaluation of 14 tumor cell lines and tissues showed similar consistently high phosphorylated peptides in their basal profiles. Incubation of four patient-derived tumor lysates with the TKIs dasatinib, sunitinib, sorafenib and erlotinib primarily caused inhibition of substrates that were highly phosphorylated in the basal profile analyses. Using recombinant Src and Axl kinase, relative substrate specificity was demonstrated for a subset of peptides, as their phosphorylation was reverted by co-incubation with a specific inhibitor. In conclusion, we demonstrated robust technical specifications of this high-throughput tyrosine kinase peptide microarray. These features required as little as 5-7 µg of protein per sample, facilitating clinical implementation as a TKI selection tool. However, currently available peptide substrates can benefit from an enhancement of the differential potential for complex samples such as tumor lysates. We propose that mass spectrometry-based phosphoproteomics may provide such an enhancement by identifying more discriminative peptides.

Differential effects of inhibitors of the PI3K/mTOR pathway on the expansion and functionality of regulatory T cells.

The PI3K/mTOR pathway is commonly deregulated in cancer. mTOR inhibitors are registered for the treatment of several solid tumors and novel inhibitors are explored clinically. Notably, this pathway also plays an important role in immunoregulation. While mTOR inhibitors block cell cycle progression of conventional T cells (Tconv), they also result in the expansion of CD4(+)CD25(hi)FOXP3(+) regulatory T cells (Tregs), and this likely limits their clinical antitumor efficacy. Here, we compared the effects of dual mTOR/PI3K inhibition (using BEZ235) to single PI3K (using BKM120) or mTOR inhibition (using rapamycin and everolimus) on Treg expansion and functionality. Whereas rapamycin, everolimus and BEZ235 effected a relative expansion benefit for Tregs and increased their overall suppressive activity, BKM120 allowed for similar expansion rates of Tregs and Tconv without altering their overall suppressive activity. Therefore, PI3K inhibition alone might offer antitumor efficacy without the detrimental selective expansion of Tregs associated with mTOR inhibition.

mTOR Inhibition Per Se Induces Nuclear Localization of FOXP3 and Conversion of Invariant NKT (iNKT) Cells into Immunosuppressive Regulatory iNKT Cells.

CD1d-restricted activation of invariant NKT (iNKT) cells results in the abundant production of various types of cytokines and the subsequent modulation of immune responses. This has been shown to be relevant in several clinical disorders, including cancer, autoimmunity, and graft tolerance. Although it is well known that the suppressive function of regulatory T cells is critically dependent on the FOXP3 gene, FOXP3 can also be expressed by conventional human T cells upon activation, indicating the lack of specificity of FOXP3 as a marker for suppressive cells. In this study, we report that the mammalian target of rapamycin (mTOR) inhibitor rapamycin and IL-10, but not TGF-ß, can induce FOXP3 expression in iNKT cell lines. Importantly, however, FOXP3(+) iNKT cells only acquired suppressive abilities when cultured in the presence of the mTOR inhibitor rapamycin. Suppression of responder T cell proliferation by FOXP3(+) iNKT cells was found to be cell contact-dependent and was accompanied by a reduced capacity of iNKT cells to secrete IFN-γ. Notably, imaging flow cytometry analysis demonstrated predominant nuclear localization of FOXP3 in suppressive FOXP3(+) iNKT cells, whereas nonsuppressive FOXP3(+) iNKT cells showed a predominance of cytoplasmically localized FOXP3. In conclusion, whereas IL-10 can enhance FOXP3 expression in iNKT cells, mTOR inhibition is solely required for promoting nuclear localization of FOXP3 and the induction of suppressive FOXP3(+) iNKT cells.

Aminobisphosphonates inhibit dendritic cell-mediated antigen-specific activation of CD1d-restricted iNKT cells.

CD1d-restricted invariant natural killer T (iNKT) cells constitute an important immunoregulatory T cell subset that can be activated by the synthetic glycolipid α-galactosylceramide (α-GalCer) and initiate antitumor immune responses. As cancer patients are frequently treated with aminobisphosphonates (NBP), it is relevant to determine possible effects of NBP on CD1d-restricted glycolipid Ag-presentation to iNKT cells. We report a striking reduction of α-GalCer-induced iNKT cell activation by monocyte derived dendritic cells (moDC) upon their exposure to NBP during maturation. We found that production of apolipoprotein E (apoE), which is a known facilitator of trans-membrane transport of exogenously derived glycolipids, was significantly diminished in moDC exposed to NBP. As the inhibitory effect of NBP on iNKT cell activation was alleviated by exogenous apoE, our data indicate that reduced apoE production by antigen presenting cells (APC) through NBP limits glycolipid-induced iNKT cell activation. This should be taken into account in the design of iNKT cell-based anti-cancer therapies.

Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate.

PURPOSE: Glutamine addiction in c-MYC-overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. EXPERIMENTAL DESIGN: A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress-mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. RESULTS: We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress-mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc-overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. CONCLUSIONS: AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA's effectiveness provide a strong rationale for further clinical development, particularly for c-MYC-overexpressing breast cancers.

Modulation of signaling enhances the efficacy of the combination of satraplatin and erlotinib.

UNLABELLED: The active metabolite (JM118) of the oral platinum analog satraplatin (JM216) was investigated for potential synergism with erlotinib, an epidermal growth factor receptor (EGFR) inhibitor. JM118 sensitivity of 7 cancer cell lines (ovarian: 2008, A2780; colon: Lovo92, WiDr; lung: A549, SW1573; epidermoid: A431), was enhanced most pronounced when JM118 preceded erlotinib, which was associated with increased formation of DNA-platinum adducts. The combination increased G2/M phase accumulation and enhanced apoptosis. JM118 increased the phosphorylation of the cell cycle proteins CDK2 and CHK1 after 24 hr exposure. JM118/erlotinib enhanced Erk and Akt phosphorylation after 2 hr. JM118 significantly decreased the phosphorylation of PTEN, VEGFR, EPHA1, ERBB4, FGF-R, andSTAT3 by 20 (PTEN) to >90% (STAT3). CONCLUSION: Erlotinib enhanced the effects of JM118, even in cells with mutations in Ras. The mechanism of synergy involved a combination of effects on platinum-DNA adduct formation, cell cycle distribution and signaling.

Phase I-II study of everolimus and low-dose oral cyclophosphamide in patients with metastatic renal cell cancer.

BACKGROUND: For patients with metastatic renal cell cancer (mRCC) who progressed on vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor therapy, the orally administered mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to prolong progression free survival. Intriguingly, inhibition of mTOR also promotes expansion of immunosuppressive regulatory T cells (Tregs) that can inhibit anti-tumor immune responses in a clinically relevant way in various tumor types including RCC. This study intends to investigate whether the antitumor efficacy of everolimus can be increased by preventing the detrimental everolimus induced expansion of Tregs using a metronomic schedule of cyclophosphamide. METHODS/DESIGN: This phase I-II trial is a national multi-center study of different doses and schedules of low-dose oral cyclophosphamide in combination with a fixed dose of everolimus in patients with mRCC not amenable to or progressive after a VEGF-receptor tyrosine kinase inhibitor containing treatment regimen. In the phase I part of the study the optimal Treg-depleting dose and schedule of metronomic oral cyclophosphamide when given in combination with everolimus will be determined. In the phase II part of the study we will evaluate whether the percentage of patients progression free at 4 months of everolimus treatment can be increased from 50% to 70% by adding metronomic cyclophosphamide (in the dose and schedule determined in the phase I part). In addition to efficacy, we will perform extensive immune monitoring with a focus on the number, phenotype and function of Tregs, evaluate the safety and feasibility of the combination of everolimus and cyclophosphamide, perform monitoring of selected angiogenesis parameters and analyze everolimus and cyclophosphamide drug levels. DISCUSSION: This phase I-II study is designed to determine whether metronomic cyclophosphamide can be used to counter the mTOR inhibitor everolimus induced Treg expansion in patients with metastatic renal cell carcinoma and increase the antitumor efficacy of everolimus. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01462214, EudraCT number 2010-024515-13, Netherlands Trial Register number NTR3085.