Myeloid-derived itaconate suppresses cytotoxic CD8+ T cells and promotes tumour growth.

The tumour microenvironment possesses mechanisms that suppress anti-tumour immunity. Itaconate is a metabolite produced from the Krebs cycle intermediate cis-aconitate by the activity of immune-responsive gene 1 (IRG1). While it is known to be immune modulatory, the role of itaconate in anti-tumour immunity is unclear. Here, we demonstrate that myeloid-derived suppressor cells (MDSCs) secrete itaconate that can be taken up by CD8+ T cells and suppress their proliferation, cytokine production and cytolytic activity. Metabolite profiling, stable-isotope tracing and metabolite supplementation studies indicated that itaconate suppressed the biosynthesis of aspartate and serine/glycine in CD8+ T cells to attenuate their proliferation and function. Host deletion of Irg1 in female mice bearing allografted tumours resulted in decreased tumour growth, inhibited the immune-suppressive activities of MDSCs, promoted anti-tumour immunity of CD8+ T cells and enhanced the anti-tumour activity of anti-PD-1 antibody treatment. Furthermore, we found a significant negative correlation between IRG1 expression and response to PD-1 immune checkpoint blockade in patients with melanoma. Our findings not only reveal a previously unknown role of itaconate as an immune checkpoint metabolite secreted from MDSCs to suppress CD8+ T cells, but also establish IRG1 as a myeloid-selective target in immunometabolism whose inhibition promotes anti-tumour immunity and enhances the efficacy of immune checkpoint protein blockade.

Labile Heme and Heme Oxygenase-1 Maintain Tumor-Permissive Niche for Endometriosis-Associated Ovarian Cancer.

Endometriosis, a painful gynecological condition accompanied by inflammation in women of reproductive age, is associated with an increased risk of ovarian cancer. We evaluated the role of peritoneal heme accumulated during menstrual cycling, as well as peritoneal and lesional macrophage phenotype, in promoting an oncogenic microenvironment. We quantified the heme-degrading enzyme, heme oxygenase-1 (HO-1, encoded by Hmox1) in normal peritoneum, endometriotic lesions and endometriosis-associated ovarian cancer (EAOC) of clear cell type (OCCC). HO-1 was expressed primarily in macrophages and increased in endometrioma and OCCC tissues relative to endometriosis and controls. Further, we compared cytokine expression profiles in peritoneal macrophages (PM) and peripheral blood mononuclear cells (PBMC) in women with endometriosis versus controls as a measure of a tumor-promoting environment in the peritoneum. We found elevated levels of HO-1 along with IL-10 and the pro-inflammatory cytokines (IL-1ß, IL-16, IFNγ) in PM but not in PBMC from endometriosis patients. Using LysM-Cre:Hmox1flfl conditional knockout mice, we show that a deficiency of HO-1 in macrophages led to the suppression of growth of ID8 ovarian tumors implanted into the peritoneum. The restriction of ID8 ovarian tumor growth was associated with an increased number of Mac3+ macrophage and B cells in LysM-Cre:Hmox1flfl mice compared to controls. Functional experiments in ovarian cancer cell lines show that HO-1 is induced by heme. Low levels of exogenous heme promoted ovarian cancer colony growth in soft agar. Higher doses of heme led to slower cancer cell colony growth in soft agar and the induction of HO-1. These data suggest that perturbation of heme metabolism within the endometriotic niche and in cancer cells themselves may be an important factor that influences tumor initiation and growth.

HO-1 and Heme: G-Quadruplex Interaction Choreograph DNA Damage Responses and Cancer Growth.

Many anti-cancer therapeutics lead to the release of danger associated pattern molecules (DAMPs) as the result of killing large numbers of both normal and transformed cells as well as lysis of red blood cells (RBC) (hemolysis). Labile heme originating from hemolysis acts as a DAMP while its breakdown products exert varying immunomodulatory effects. Labile heme is scavenged by hemopexin (Hx) and processed by heme oxygenase-1 (HO-1, Hmox1), resulting in its removal and the generation of biliverdin/bilirubin, carbon monoxide (CO) and iron. We recently demonstrated that labile heme accumulates in cancer cell nuclei in the tumor parenchyma of Hx knockout mice and contributes to the malignant phenotype of prostate cancer (PCa) cells and increased metastases. Additionally, this work identified Hx as a tumor suppressor gene. Direct interaction of heme with DNA G-quadruplexes (G4) leads to altered gene expression in cancer cells that regulate transcription, recombination and replication. Here, we provide new data supporting the nuclear role of HO-1 and heme in modulating DNA damage response, G4 stability and cancer growth. Finally, we discuss an alternative role of labile heme as a nuclear danger signal (NDS) that regulates gene expression and nuclear HO-1 regulated DNA damage responses stimulated by its interaction with G4.

Therapeutic Repurposing of Biguanides in Cancer.

Biguanides are a class of antidiabetic drugs that includes phenformin and metformin; however, the former was withdrawn from approval in many countries due to its toxicity. Findings from retrospective epidemiological studies in diabetic populations and preclinical laboratory models have demonstrated that biguanides possess antitumor activities that suggest their repurposing for cancer prevention and treatment. However, a better understanding of how these biguanides behave as antitumor agents is needed to guide their improved applications in cancer therapy, spurring increased interest in their pharmacology. Here, we present evidence for proposed mechanisms of action related to their antitumor activity, including their effects on central carbon metabolism in cancer cells and immune-modulating activity, and then review progress on biguanide repurposing in cancer therapeutics and the possible re-evaluation of phenformin as a cancer therapeutic agent.

Heme-Derived Metabolic Signals Dictate Immune Responses.

Heme is one of the most abundant molecules in the body acting as the functional core of hemoglobin/myoglobin involved in the O2/CO2 carrying in the blood and tissues, redox enzymes and cytochromes in mitochondria. However, free heme is toxic and therefore its removal is a significant priority for the host. Heme is a well-established danger-associated molecular pattern (DAMP), which binds to toll-like receptor 4 (TLR4) to induce immune responses. Heme-derived metabolites including the bile pigments, biliverdin (BV) and bilirubin (BR), were first identified as toxic drivers of neonatal jaundice in 1800 but have only recently been appreciated as endogenous drivers of multiple signaling pathways involved in protection from oxidative stress and regulators of immune responses. The tissue concentration of heme, BV and BR is tightly controlled. Heme oxygenase-1 (HO-1, encoded by HMOX1) produces BV by heme degradation, while biliverdin reductase-A (BLVR-A) generates BR by the subsequent conversion of BV. BLVR-A is a fascinating protein that possesses a classical protein kinase domain, which is activated in response to BV binding to its enzymatic site and initiates the downstream mitogen-activated protein kinases (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. This links BLVR-A activity to cell growth and survival pathways. BLVR-A also contains a bZip DNA binding domain and a nuclear export sequence (NES) and acts as a transcription factor to regulate the expression of immune modulatory genes. Here we will discuss the role of heme-related immune response and the potential for targeting the heme system for therapies directed toward hepatitis and cancer.

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma.

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% with current standard of care. Tumors often recur within 9 months following initial surgery, radiation and chemotherapy, at which point treatment options become limited. This highlights the pressing need for the development of better therapeutics to prolong survival and increase the quality of life for these patients. Tumor Treating Fields (TTFields) therapy was developed to take advantage of the effect of low frequency alternating electrical fields on cells for cancer therapy. TTFields have been demonstrated to disrupt cells during mitosis and slow tumor growth. There is also growing evidence that they act through stimulating immune responses within exposed tumors. The advantages of TTFields therapy include its noninvasive approach and increased quality of life compared to other treatment modalities such as cytotoxic chemotherapies. The Food and Drug Administration approved TTFields therapy for the treatment of recurrent glioblastoma in 2011 and for newly diagnosed glioblastoma in 2015. We report on the effects of TTFields during mitosis, the results of electric fields modeling, and proper transducer array placement. Our protocol outlines the clinical application of TTFields on a patient post-surgery, using the second-generation device.

Reproducing the human mucosal environment ex vivo: inflammatory bowel disease as a paradigm.

PURPOSE OF REVIEW: The medical management of inflammatory bowel disease (IBD) remains problematic with a pressing need for innovation in drug development as well as delivery of personalized therapies. Both the disease's inherent pathophysiologic complexity and heterogeneity in its etiology conspire in making it difficult to accurately model for either the purposes of basic research or drug development. Multiple attempts at creating meaningful experimental models have fallen short of adequately recapitulating the disease and most do not capture any aspect of the cause or the effects of patient heterogeneity that underlays most of the difficulties faced by physicians and their patients. In vivo animal models, tissue culture systems, and more recent synthetic biology approaches are all too simplistically reductionist for the task. However, ex vivo culture platforms utilizing patient biopsies offer a system that more closely mimics end-stage disease processes that can be studied in detail and subjected to experimental manipulations. RECENT FINDINGS: Recent studies describe further optimization of mucosal explant cultures in order to increase tissue viability and maintain a polarized epithelial layer. Current applications of the platform include studies of the interplay between the epithelial, immune and stromal compartment of the intestinal tissue, investigation of host-microbial interactions, preclinical evaluation of candidate drugs and uncovering mechanisms of action of established or emerging treatments for IBD. SUMMARY: Patient explant-based assays offer an advanced biological system in IBD that recapitulates disease complexity and reflects the heterogeneity of the patient population. In its current stage of development, the system can be utilized for drug testing prior to the costlier and time-consuming evaluation by clinical trials. Further refinement of the technology and establishment of assay readouts that correlate with therapeutic outcomes will yield a powerful tool for personalized medicine approaches in which individual patient responses to available treatments are assessed a priori, thus reducing the need for trial and error within the clinical setting.

Alternating Electric Fields Therapy for Malignant Gliomas: From Bench Observation to Clinical Reality.

Alternating electric fields of intermediate frequencies, also known as Tumor Treating Fields (TTFields or TTF) is a novel anticancer treatment modality that disrupts tumor cell mitosis at the metaphase-anaphase transition, leading to mitotic catastrophe, aberrant mitotic exit, and/or cell death. It is realized through alteration of the cytokinetic cleavage furrow by interference of proteins possessing large dipole moments, like septin heterotrimer complex and α/ß-tubulin, and that results in disordered membrane contraction and failed cytokinesis. Aberrant mitotic exit also elicits immunogenic cell death, which may potentiate an immune response against treated tumors. Notably, in patients with recurrent glioblastoma multiforme (GBM) a prospective clinical trial demonstrated comparable overall survival and progression-free survival after TTFields therapy and best physician's choice chemotherapy. Moreover, it was shown that in patients with newly diagnosed GBM initially treated with standard chemoradiotherapy with daily temozolomide (TMZ), adjuvant TTFields combined with TMZ offered better survival than adjuvant TMZ alone. Therefore, TTFields therapy can be appreciated as a standard treatment option in cases of intracranial malignant gliomas, whereas future studies should establish its optimal combination with other existing anticancer modalities, which may offer additional survival benefits for patients.

Supplementing Cancer?

In this issue of Molecular Cell, Lin et al. (2018) report that chondroitin-4-sulfate, which is found in a common supplement meant to alleviate degenerative joint disorders, promotes the growth of BRAF V600E mutant melanoma. This study not only has implications for patient care but also sheds light on a novel mechanism for regulating phosphoinositide 3-kinase signaling.

Literature Review of Spinal Cord Glioblastoma.

OBJECTIVES: This systematic review aims to investigate spinal cord glioblastoma (scGBM) and correlations between patient traits and survival outcome, as well as differences in cohorts administered temozolomide or total resections, through an analysis of published cases reported up to October 2016. METHODS: We obtained patient data by querying PubMed and Google Scholar with predetermined search terms and inclusion criteria that enabled the identification of relevant case reports. Survival was compared using Kaplan-Meier curves and log-rank analyses. RESULTS: Of 153 patients with scGBM identified through a literature search, 135 met the predetermined search and inclusion criteria. Median overall survival (OS) for the resulting cohort was 12 (95% CI, 10-14) months. The female sex was found to significantly predict worse outcomes, and a sizable number of patients with long-term disease were found to have afflictions of the thoracic spinal cord. Neither the pediatric, temozolomide nor total resection subgroups had significantly improved survival characteristics, by log-rank analysis, relative to counterparts. CONCLUSIONS: These data elucidate the characteristics of patients with scGBM. For more sophisticated and in-depth analyses in the future, it is imperative that time-of-treatment information is recorded in future case reports. In addition, all case reports should be made available to prevent publication bias.

ZNF143 protein is an important regulator of the myeloid transcription factor C/EBPα.

The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, ∼100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.

Phenformin Inhibits Myeloid-Derived Suppressor Cells and Enhances the Anti-Tumor Activity of PD-1 Blockade in Melanoma.

Biguanides, such as the diabetes therapeutics metformin and phenformin, have shown antitumor activity both in vitro and in vivo. However, their potential effects on the tumor microenvironment are largely unknown. Here we report that phenformin selectively inhibits granulocytic myeloid-derived suppressor cells in spleens of tumor-bearing mice and ex vivo. Phenformin induces production of reactive oxygen species in granulocytic myeloid-derived suppressor cells, whereas the antioxidant N-acetylcysteine attenuates the inhibitory effects of phenformin. Co-treatment of phenformin enhances the effect of anti-PD-1 antibody therapy on inhibiting tumor growth in the BRAF V600E/PTEN-null melanoma mouse model. Combination of phenformin and anti PD-1 cooperatively induces CD8+ T-cell infiltration and decreases levels of proteins that are critical for immune suppressive activities of myeloid-derived suppressor cells. Our findings show a selective, inhibitory effect of phenformin on granulocytic myeloid-derived suppressor cell-driven immune suppression and support that phenformin improves the anti-tumor activity of PD-1 blockade immunotherapy in melanoma.

Cabozantinib Eradicates Advanced Murine Prostate Cancer by Activating Antitumor Innate Immunity.

Several kinase inhibitors that target aberrant signaling pathways in tumor cells have been deployed in cancer therapy. However, their impact on the tumor immune microenvironment remains poorly understood. The tyrosine kinase inhibitor cabozantinib showed striking responses in cancer clinical trial patients across several malignancies. Here, we show that cabozantinib rapidly eradicates invasive, poorly differentiated PTEN/p53-deficient murine prostate cancer. This was associated with enhanced release of neutrophil chemotactic factors from tumor cells, including CXCL12 and HMGB1, resulting in robust infiltration of neutrophils into the tumor. Critically, cabozantinib-induced tumor clearance in mice was abolished by antibody-mediated granulocyte depletion or HMGB1 neutralization or blockade of neutrophil chemotaxis with the CXCR4 inhibitor plerixafor. Collectively, these data demonstrate that cabozantinib triggers a neutrophil-mediated anticancer innate immune response, resulting in tumor clearance.Significance: This study is the first to demonstrate that a tyrosine kinase inhibitor can activate neutrophil-mediated antitumor innate immunity, resulting in invasive cancer clearance. Cancer Discov; 7(7); 750-65. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 653.

An Overview of Alternating Electric Fields Therapy (NovoTTF Therapy) for the Treatment of Malignant Glioma.

As with many cancer treatments, tumor treating fields (TTFields) target rapidly dividing tumor cells. During mitosis, TTFields-exposed cells exhibit uncontrolled membrane blebbing at the onset of anaphase, resulting in aberrant mitotic exit. Based on these criteria, at least two protein complexes have been proposed as TTFields' molecular targets, including α/ß-tubulin and the septin 2, 6, 7 heterotrimer. After aberrant mitotic exit, cells exhibited abnormal nuclei and signs of cellular stress, including decreased cellular proliferation and p53 dependence, and exhibit the hallmarks of immunogenic cell death, suggesting that TTFields treatment may induce an antitumor immune response. Clinical trials lead to Food and Drug Administration approval for their treatment of recurrent glioblastoma. Detailed modeling of TTFields within the brain suggests that the location of the tumor may affect treatment efficacy. These observations have a profound impact on the use of TTFields in the clinic, including what co-therapies may be best applied to boost its efficacy.

Tumor treating fields therapy device for glioblastoma: physics and clinical practice considerations.

Alternating electric fields therapy, as delivered by the tumor treating fields device, is a new modality of cancer treatment that has been approved by the US FDA for recurrent glioblastoma. At a frequency of 200 kHz, these fields emanate from transducer arrays on the surface of the patient's scalp into the brain and perturb processes necessary for cytokinesis during tumor cell mitosis. In the registration Phase III trial for recurrent glioblastoma patients, the efficacy of the tumor treating fields as monotherapy was equivalent to chemotherapy, while scalp irritation was its major adverse event compared with systemic toxicities that were associated with cytotoxic chemotherapies. Alternating electric fields therapy is, therefore, an essential option for the treatment of recurrent glioblastoma. Here, we summarize our current knowledge of the physics, cell biology and clinical data supporting the use of the tumor treating fields therapy.

An Evidence-Based Review of Alternating Electric Fields Therapy for Malignant Gliomas.

OPINION STATEMENT: Glioblastoma is a deadly disease and even aggressive neurosurgical resection followed by radiation and chemotherapy only extends patient survival to a median of 1.5 years. The challenge in treating this type of tumor stems from the rapid proliferation of the malignant glioma cells, the diffuse infiltrative nature of the disease, multiple activated signal transduction pathways within the tumor, development of resistant clones during treatment, the blood brain barrier that limits the delivery of drugs into the central nervous system, and the sensitivity of the brain to treatment effect. Therefore, new therapies that possess a unique mechanism of action are needed to treat this tumor. Recently, alternating electric fields, also known as tumor treating fields (TTFields), have been developed for the treatment of glioblastoma. TTFields use electromagnetic energy at an intermediate frequency of 200 kHz as a locoregional intervention and act to disrupt tumor cells as they undergo mitosis. In a phase III clinical trial for recurrent glioblastoma, TTFields were shown to have equivalent efficacy when compared to conventional chemotherapies, while lacking the typical side effects associated with chemotherapies. Furthermore, an interim analysis of a recent clinical trial in the upfront setting demonstrated superiority to standard of care cytotoxic chemotherapy, most likely because the subjects' tumors were at an earlier stage of clonal evolution, possessed less tumor-induced immunosuppression, or both. Therefore, it is likely that the efficacy of TTFields can be increased by combining it with other anti-cancer treatment modalities.

Tumor treating fields perturb the localization of septins and cause aberrant mitotic exit.

The anti-tumor effects of chemotherapy and radiation are thought to be mediated by triggering G1/S or G2/M cell cycle checkpoints, while spindle poisons, such as paclitaxel, block metaphase exit by initiating the spindle assembly checkpoint. In contrast, we have found that 150 kilohertz (kHz) alternating electric fields, also known as Tumor Treating Fields (TTFields), perturbed cells at the transition from metaphase to anaphase. Cells exposed to the TTFields during mitosis showed normal progression to this point, but exhibited uncontrolled membrane blebbing that coincided with metaphase exit. The ability of such alternating electric fields to affect cellular physiology is likely to be dependent on their interactions with proteins possessing high dipole moments. The mitotic Septin complex consisting of Septin 2, 6 and 7, possesses a high calculated dipole moment of 2711 Debyes (D) and plays a central role in positioning the cytokinetic cleavage furrow, and governing its contraction during ingression. We showed that during anaphase, TTFields inhibited Septin localization to the anaphase spindle midline and cytokinetic furrow, as well as its association with microtubules during cell attachment and spreading on fibronectin. After aberrant metaphase exit as a consequence of TTFields exposure, cells exhibited aberrant nuclear architecture and signs of cellular stress including an overall decrease in cellular proliferation, followed by apoptosis that was strongly influenced by the p53 mutational status. Thus, TTFields are able to diminish cell proliferation by specifically perturbing key proteins involved in cell division, leading to mitotic catastrophe and subsequent cell death.