Unveiling the epigenomic mechanisms of acquired platinum-resistance in high-grade serous ovarian cancer.

Resistance to platinum-based chemotherapy is the major cause of death from high-grade serous ovarian cancer (HGSOC). We hypothesise that detection of specific DNA methylation changes may predict platinum resistance in HGSOC. Using a publicly available "discovery" dataset we examined epigenomic and transcriptomic alterations between primary platinum-sensitive (n = 32) and recurrent acquired drug resistant HGSOC (n = 28) and identified several genes involved in immune and chemoresistance-related pathways. Validation via high-resolution melt analysis of these findings, in cell lines and HGSOC tumours, demonstrated the most consistent changes were observed in three of the genes: APOBEC3A, NKAPL and PDCD1. Plasma samples from an independent HGSOC cohort (n = 17) were analysed using droplet digital PCR. Hypermethylation of NKAPL was detected in 46% and hypomethylation of APOBEC3A in 69% of plasma samples taken from women with relapsed HGSOC (n = 13), with no alterations identified in disease-free patients (n = 4). Following these results, and using a CRISPR-Cas9 approach, we were also able to demonstrate that in vitro NKAPL promoter demethylation increased platinum sensitivity by 15%. Overall, this study demonstrates the importance of aberrant methylation, especially of the NKAPL gene, in acquired platinum resistance in HGSOC.

γδ T cell dichotomy with opposing cytotoxic and wound healing functions in human solid tumors.

γδ T cells are important tissue-resident, innate T cells that are critical for tissue homeostasis. γδ cells are associated with positive prognosis in most tumors; however, little is known about their heterogeneity in human cancers. Here, we phenotyped innate and adaptive cells in human colorectal (CRC) and endometrial cancer. We found striking differences in γδ subsets and function in tumors compared to normal tissue, and in the γδ subsets present in tumor types. In CRC, an amphiregulin (AREG)-producing subset emerges, while endometrial cancer is infiltrated by cytotoxic cells. In humanized CRC models, tumors induced this AREG phenotype in Vδ1 cells after adoptive transfer. To exploit the beneficial roles of γδ cells for cell therapy, we developed an expansion method that enhanced cytotoxic function and boosted metabolic flexibility, while eliminating AREG production, achieving greater tumor infiltration and tumor clearance. This method has broad applications in cellular therapy as an 'off-the-shelf' treatment option.

Heightened metabolic responses in NK cells from patients with neuroblastoma suggests increased potential for immunotherapy.

High risk neuroblastoma is responsible for 15% of deaths in pediatric cancer patients. The introduction of anti-GD2 immunotherapy has significantly improved outcomes but there is still only approximately a 50% 5 year event-free-survival for these children and improvements in treatments are urgently required. Anti-GD2 immunotherapy uses the patients' own immune system to kill cancer cells. In particular, Natural Killer (NK) cells kill antibody coated tumor cells by a process called antibody dependent cellular cytotoxicity (ADCC). However, our previous work has highlighted metabolic exhaustion of NK cells in circulating blood of adult cancer patients, identifying this as a potential therapeutic target. In this study, we investigated circulating NK cells in patients newly diagnosed with neuroblastoma. We found evidence of activation of NK cells in vivo by the cancer itself. While some evidence of NK cell dysfunction was observed in terms of IFNγ production, most results indicated that the NK cell compartment remained relatively intact. In fact, some aspects of metabolic and functional activities were actually increased in patients compared to controls. Glycolytic responses, which we show are crucial for ADCC, were actually enhanced in patients and CD16, the NK cell receptor that mediates ADCC, was also expressed at high levels in some patients. Overall, the data suggest that patient NK cells could be harvested at diagnosis for subsequent beneficial autologous use during immunotherapy. Enhancing glycolytic capacity of cell therapies could also be a strategic goal of future cell therapies for patients with neuroblastoma and indeed other cancers.

TGFß drives NK cell metabolic dysfunction in human metastatic breast cancer.

BACKGROUND: Natural killer (NK) cells provide important immune protection from cancer and are a key requirement for particular immunotherapies. There is accumulating evidence that NK cells become dysfunctional during cancer. Overcoming NK cell exhaustion would be an important step to allow them to function optimally in a range of NK cell therapies, including those that depend on autologos circulating NK cells. We have previously demonstrated that NK cells undergo a normal metabolic reprogramming in response to cytokine activation and that this is required for optimal function. The objective of this work was to investigate if cellular metabolism of circulating NK cells is dysregulated in patients with metastatic breast cancer and if so, to gain insights into potential mechanisms underpinning this. Such discoveries would provide important insights into how to unleash the full activity of NK cells for maximum immunotherapy output. METHODS: Single-cell analysis, metabolic flux and confocal analysis of NK cells from patients with metastatic breast cancer and healthy controls RESULTS: In addition to reduced interferon-γ production and cytotoxicity, peripheral blood NK cells from patients had clear metabolic deficits including reduced glycolysis and oxidative phosphorylation. There were also distinct morphologically alterations in the mitochondria with increased mitochondrial fragmentation observed. Transforminggrowth factor-ß (TGFß) was identified as a key driver of this phenotype as blocking its activity reversed many metabolic and functional readouts. Expression of glycoprotein-A repetitions predominant (GARP) and latency associated peptide (LAP), which are involved with a novel TGFß processing pathway, was increased on NK cells from some patients. Blocking the GARP-TGFß axis recapitulated the effects of TGFß neutralization, highlighting GARP as a novel NK cell immunotherapy target for the first time. CONCLUSIONS: TGFß contributes to metabolic dysfunction of circulating NK cells in patients with metastatic breast cancer. Blocking TGFß and/or GARP can restore NK cell metabolism and function and is an important target for improving NK cell-based immunotherapies.

Membrane-bound HSP70-engineered myeloma cell-derived exosomes stimulate more efficient CD8(+) CTL- and NK-mediated antitumour immunity than exosomes released from heat-shocked tumour cells expressing cytoplasmic HSP70.

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour-derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T-cell immune responses. To enhance EXO-based antitumour immunity, we generated an engineered myeloma cell line J558(HSP) expressing endogenous P1A tumour antigen and transgenic form of membrane-bound HSP70 and heat-shocked J558(HS) expressing cytoplasmic HSP70, and purified EXO(HSP) and EXO(HS) from J558(HSP) and J558(HS) tumour cell culture supernatants by ultracentrifugation. We found that EXO(HSP) were able to more efficiently stimulate maturation of DCs with up-regulation of Ia(b) , CD40, CD80 and inflammatory cytokines than EXO(HS) after overnight incubation of immature bone-marrow-derived DCs (5 × 106 cells) with EXO (100 µg), respectively. We also i.v. immunized BALB/c mice with EXO (30 µg/mouse) and assessed P1A-specific T-cell responses after immunization. We demonstrate that EXO(HSP) are able to stimulate type 1 CD4(+) helper T (Th1) cell responses, and more efficient P1A-specific CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXO(HS) . In addition, we further elucidate that EXO(HSP) -stimulated antitumour immunity is mediated by both P1A-specific CD8(+) CTL and non-P1A-specific natural killer (NK) responses. Therefore, membrane-bound HSP70-expressing tumour cell-released EXO may represent a more effective EXO-based vaccine in induction of antitumour immunity.

NK Cell Metabolism and TGFß - Implications for Immunotherapy.

NK cells are innate lymphocytes which play an essential role in protection against cancer and viral infection. Their functions are dictated by many factors including the receptors they express, cytokines they respond to and changes in the external environment. These cell processes are regulated within NK cells at many levels including genetic, epigenetic and expression (RNA and protein) levels. The last decade has revealed cellular metabolism as another level of immune regulation. Specific immune cells adopt metabolic configurations that support their functions, and this is a dynamic process with cells undergoing metabolic reprogramming during the course of an immune response. Upon activation with pro-inflammatory cytokines, NK cells upregulate both glycolysis and oxphos metabolic pathways and this supports their anti-cancer functions. Perturbation of these pathways inhibits NK cell effector functions. Anti-inflammatory cytokines such as TGFß can inhibit metabolic changes and reduce functional outputs. Although a lot remains to be learned, our knowledge of potential molecular mechanisms involved is growing quickly. This review will discuss our current knowledge on the role of TGFß in regulating NK cell metabolism and will draw on a wider knowledge base regarding TGFß regulation of cellular metabolic pathways, in order to highlight potential ways in which TGFß might be targeted to contribute to the exciting progress that is being made in terms of adoptive NK cell therapies for cancer.

The XIAP inhibitor embelin sensitises malignant rhabdoid tumour cells to TRAIL treatment via enhanced activation of the extrinsic apoptotic pathway.

Malignant rhabdoid tumour (MRT) is a rare, aggressive paediatric neoplasm, primarily diagnosed in those below the age of three. MRTs most commonly arise in the central nervous system and kidneys. A poor prognosis accompanies the MRT diagnosis, with a reported 2­year survival rate of 30%. Thus, there is an urgent need for the development of new therapies for this malignancy. Members of the inhibitor of apoptosis protein (IAP) family have previously been reported to be overexpressed in various cancers. As such, small molecule inhibitors of these family members have entered clinical trials. However, the role of IAPs in MRT has not been examined yet. The present study is the first report of the expression of a range of IAPs, including X­linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1), cellular inhibitor of apoptosis protein 2 (cIAP2), livin and survivin in MRT cell lines. Furthermore, the results demonstrated the ability of the XIAP inhibitor, embelin, to sensitise MRT cell lines to TNF­related apoptosis­inducing ligand (TRAIL) treatment. The enhanced cell death detected upon cotreatment was dependent on caspase­8 and co­occurred with caspase­8 and caspase­3 cleavage, suggesting engagement of the extrinsic apoptotic pathway. Sensitisation to TRAIL was accompanied by livin cleavage, alongside downregulation of survivin and the caspase­8 inhibitor FLIPL. In addition, knockdown of XIAP using siRNA enhanced TRAIL­mediated cell death, suggesting that this process may in part mediate sensitisation. In conclusion, the present results suggested that IAP inhibition may present a novel avenue for the treatment of MRT.

Tumor apoptotic bodies inhibit CTL responses and antitumor immunity via membrane-bound transforming growth factor-beta1 inducing CD8+ T-cell anergy and CD4+ Tr1 cell responses.

Tumor cell apoptosis induced by radiation therapy results in apoptotic tumor cells and apparition of membrane blebs termed apoptotic bodies (APB). The immune responses induced by apoptotic tumor cells have been extensively studied. However, the role of APB in modulation of tumor immune responses is elusive. In this study, we induced apoptosis in 90% ovabumin-expressing EG7 tumor cells by in vitro irradiation (9,000 rad) of tumor cells with a subsequent cell culture for 9 hours. APB purified from irradiation-induced apoptotic EG7 cell culture supernatant by differential ultracentrifugation were vesicles with 50 to 90 nm in diameter and expressed apoptosis-specific Annexin V, 14-3-3, and Histone H3. We then investigated its potential modulation in DC(OVA)-induced T-cell responses and antitumor immunity. We found that EG7-derived APB were tolerogenic and capable of suppressing DC(OVA)-stimulated CD8+ CTL responses and antitumor immunity via its induction of CD8+ T-cell anergy and type 1 regulatory CD4+ T-cell responses. Analysis of apoptotic tumor cells and APB revealed the expression of membrane-bound transforming growth factor (TGF)-beta1 associated with irradiation-induced apoptosis formation, which is a result from activation of transcriptional factor NF-AT specific for TGF-beta1 promoters. Our data further elucidate that it is the membrane-bound TGF-beta1 expression on APB that contributes to its in vitro antiproliferative effect as shown by using neutralizing TGF-beta1-specific antibody. Administration of anti-TGF-beta1 antibody in vivo also blocked APB-mediated immune suppression of CD8+ CTL responses and antitumor immunity. Therefore, our study may have great impact in designing a combined radiation therapy with immunotherapy of cancer.