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Cancer cells take up amino acids from the extracellular space to drive cell proliferation and viability. Similar mechanisms are applied by immune cells, resulting in the competition between conventional T cells, or indeed chimeric antigen receptor (CAR) T cells and tumor cells, for the limited availability of amino acids within the environment. We demonstrate that T cells can be re-engineered to express SLC7A5 or SLC7A11 transmembrane amino acid transporters alongside CARs. Transporter modifications increase CAR T-cell proliferation under low tryptophan or cystine conditions with no loss of CAR cytotoxicity or increased exhaustion. Transcriptomic and phenotypic analysis reveals that downstream, SLC7A5/SLC7A11–modified CAR T cells upregulate intracellular arginase expression and activity. In turn, we engineer and phenotype a further generation of CAR T cells that express functional arginase 1/arginase 2 enzymes and have enhanced CAR T-cell proliferation and antitumor activity. Thus, CAR T cells can be adapted to the amino acid metabolic microenvironment of cancer, a hitherto recognized but unaddressed barrier for successful CAR T-cell therapy.

Supplementary Materials and Methods CLEAN version

Supplementary Figure 3 Neuroblastoma conditioning upregulates IL-1ï�¢ï€ and TNF-ï�¡ï€ expression in macrophages

Supplementary Figure Legends and Tables

Supplementary Figure 4 Macrophage infiltration and downstream effects on neuroblastoma tumours

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)–dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1β and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)–dependent manner. IL1β and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest–derived cells. Proteomic analysis revealed that enrichment of IL1β and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited.Significance:These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Supplementary Figure 2 Cytokine profile of tumour induced macrophages or granulocytes

Supplementary Figure 1: M1-Macrophage infiltrate neuroblastoma

Radiotherapy is a valuable treatment in the management algorithm of pituitary adenomas and craniopharyngiomas. However, the risk of second brain tumour following radiotherapy is a major concern. We assessed this risk using non-irradiated patients with the same primary pathology and imaging surveillance as controls.In this multicentre, retrospective cohort study, 4292 patients with pituitary adenoma or craniopharyngioma were identified from departmental registries at six adult endocrine centres (Birmingham, Oxford, Leeds, Leicester, and Bristol, UK and Ferrara, Italy). Patients with insufficient clinical data, known genetic predisposition to or history of brain tumour before study entry (n=532), and recipients of proton beam or stereotactic radiotherapy (n=81) were excluded. Data were analysed for 996 patients exposed to 2-dimensional radiotherapy, 3-dimensional conformal radiotherapy, or intensity-modulated radiotherapy, and compared with 2683 controls.Over 45 246 patient-years, second brain tumours were reported in 61 patients (seven malignant [five radiotherapy, two controls], 54 benign [25 radiotherapy, 29 controls]). Radiotherapy exposure and older age at pituitary tumour detection were associated with increased risk of second brain tumour. Rate ratio for irradiated patients was 2·18 (95% CI 1·31-3·62, p0·0001). Cumulative probability of second brain tumour was 4% for the irradiated and 2·1% for the controls at 20 years.Irradiated adults with pituitary adenoma or craniopharyngioma are at increased risk of second brain tumours, although this risk is considerably lower than previously reported in studies using general population controls with no imaging surveillance. Our data clarify an important clinical question and guide clinicians when counselling patients with pituitary adenoma or craniopharyngioma on the risks and benefits of radiotherapy.Pfizer.

Diffuse intrinsic pontine glioma remains a devastating condition with a dismal five year survival rate less than 5%. New approaches for treating this aggressive disease are critical to driving progress. Conventional radiotherapy remains the cornerstone of treatment, with no chemotherapeutic agent found to improve survival. However, radiotherapy is often delivered as a palliative treatment, and disease often recurs 3-6 months after. Radiation causes DNA damage and oxidative stress yielding a senescent state of replicative arrest in susceptible cells. However, increasing evidence demonstrates malignant cells can escape senescence leading to tumour recurrence. Targeted ablation of non-replicating senescent tumour cells following radiation could negate tumour recurrence. It remains unknown whether DIPG undergoes senescence following radiation, and furthermore, whether senolytics can be utilised to target senescent DIPG cells. We employed radiation to induce a senescent state in primary human DIPG cell lines. Senescence was confirmed using SA-β-gal staining, lack of EdU incorporation and qRT-PCR to characterise the SASP in three primary human DIPG cell lines. RNA-sequencing on DIPG cells following radiation revealed senescence and SASP signatures. Likewise, expression of senescence markers has been detected in human tumours. Viable cells that survive radiation were then utilised to screen candidate senolytic drugs, only Bcl-XL inhibitors demonstrated reproducible senolytic activity in radiation treated DIPG cells. In addition, Bcl-XL degradation using PROTACs (proteolysis targeting chimeras) resulted in a significant increase in senolysis of susceptible tumour cells. Conversely, Bcl-2 inhibitors failed to show any consistent senolytic activity. We are currently performing preclinical studies in the mouse to test the efficiency of senolytics against DIPG. These results demonstrate future possibilities of targeting radiation induced senescence in DIPG, using novel senolytic therapies and highlight Bcl-XL dependency as a potential vulnerability of surviving DIPG cells following exposure to radiation.

// Ashley Vardon 1, * , Madhumita Dandapani 1, * , Daryl Cheng 1, * , Paul Cheng 2 , Carmela De Santo 1 and Francis Mussai 1 1 Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham, United Kingdom 2 Bio-Cancer Treatment International Ltd, Hong Kong, China * These authors have contributed equally to this work Correspondence to: Francis Mussai, email: Francis.mussai@bch.nhs.uk Keywords: paediatric, sarcomas, brain, arginine, auxotrophism Received: October 22, 2016 Accepted: June 05, 2017 Published: June 29, 2017 ABSTRACT Paediatric sarcomas and brain tumours, remain cancers of significant unmet need, with a poor prognosis for patients with high risk disease or those who relapse, and significant morbidities from treatment for those that survive using standard treatment approaches. Novel treatment strategies, based on the underlying tumour biology, are needed to improve outcomes. Arginine is a semi-essential amino acid that is imported from the extracellular microenvironment or recycled from intracellular precursors through the combined expression of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) enzymes. The failure to express at least one of these recycling enzymes makes cells reliant on extracellular arginine – a state known as arginine auxotrophism. Here we show in large in silico patient cohorts that paediatric sarcomas and brain tumours express predominately the arginine transporter SLC7A1 and the arginine metabolising enzyme Arginase 2 (ARG2), but have low-absent expression of OTC. The arginine metabolic pathway correlated with the expression of genes associated with tumour pathogenesis, and overall survival in paediatric sarcomas. This gene signature of arginine auxotrophism indicates paediatric sarcomas and brain tumours are a viable target for therapeutic arginase drugs under current clinical trial development.

Diffuse intrinsic pontine glioma remains a devastating condition with a dismal five year survival rate less than 5%. New approaches for treating this aggressive disease are critical to driving progress. Conventional radiotherapy remains the cornerstone of treatment, with no chemotherapeutic agent found to improve survival. However, radiotherapy is often delivered as a palliative treatment, and disease often recurs 3–6 months after. Radiation causes DNA damage and oxidative stress yielding a senescent state of replicative arrest in susceptible cells. However, increasing evidence demonstrates malignant cells can escape senescence leading to tumour recurrence. Targeted ablation of non-replicating senescent tumour cells following radiation could negate tumour recurrence. It remains unknown whether DIPG undergoes senescence following radiation, and furthermore, whether senolytics can be utilised to target senescent DIPG cells. We employed radiation to induce a senescent state in primary human DIPG cell lines. Senescence was confirmed using SA-β-gal staining, lack of EdU incorporation and qRT-PCR to characterise the SASP in three primary human DIPG cell lines. RNA-sequencing on DIPG cells following radiation revealed senescence and SASP signatures. Viable cells that survive radiation were then utilised to screen candidate senolytic drugs, only Bcl-XL inhibitors demonstrated reproducible senolytic activity in radiation treated DIPG cells. Conversely, Bcl-2 inhibitors failed to show any consistent senolytic activity. These results demonstrate future possibilities of targeting radiation induced senescence in DIPG, using novel senolytic therapies and highlight Bcl-XL dependency as a potential vulnerability of surviving DIPG cells following exposure to radiation.

Renewed interest in the use of therapeutic enzymes combined with an improved knowledge of cancer cell metabolism, has led to the translation of several arginine depletion strategies into early phase clinical trials. Arginine auxotrophic tumors are reliant on extracellular arginine, due to the downregulation of arginosuccinate synthetase or ornithine transcarbamylase-key enzymes for intracellular arginine recycling. Engineered arginine catabolic enzymes such as recombinant human arginase (rh-Arg1-PEG) and arginine deiminase (ADI-PEG) have demonstrated cytotoxicity against arginine auxotrophic tumors. In this review, we discuss the molecular events triggered by extracellular arginine depletion that contribute to tumor cell death.

Neuroblastoma is the most common childhood solid tumor, yet the prognosis for high-risk disease remains poor. We demonstrate here that arginase 2 (ARG2) drives neuroblastoma cell proliferation via regulation of arginine metabolism. Targeting arginine metabolism, either by blocking cationic amino acid transporter 1 (CAT-1)–dependent arginine uptake in vitro or therapeutic depletion of arginine by pegylated recombinant arginase BCT-100, significantly delayed tumor development and prolonged murine survival. Tumor cells polarized infiltrating monocytes to an M1-macrophage phenotype, which released IL1β and TNFα in a RAC-alpha serine/threonine-protein kinase (AKT)–dependent manner. IL1β and TNFα established a feedback loop to upregulate ARG2 expression via p38 and extracellular regulated kinases 1/2 (ERK1/2) signaling in neuroblastoma and neural crest–derived cells. Proteomic analysis revealed that enrichment of IL1β and TNFα in stage IV human tumor microenvironments was associated with a worse prognosis. These data thus describe an immune-metabolic regulatory loop between tumor cells and infiltrating myeloid cells regulating ARG2, which can be clinically exploited. Significance: These findings illustrate that cross-talk between myeloid cells and tumor cells creates a metabolic regulatory loop that promotes neuroblastoma progression.

Craniopharyngiomas are epithelial tumours in the sellar/parasellar area accounting for 2–5% of all primary intracranial neoplasms and for up to 15% of intracranial tumours in children. The adamantinomatous histological subtype is the most common and the majority of them (94–95%) have a suprasellar component. Their potential proximity to, and the subsequent pressure effects on vital structures of the brain (visual pathways, brain parenchyma, ventricular system, major blood vessels and hypothalamo-pituitary system) can result to multiple clinical manifestations, the severity of which depends on the location, size and growth potential of the tumour. The main manifestations at diagnosis are neurological, visual and hypothalamo-pituitary with headaches, nausea/vomiting, visual disturbances, growth failure (in children) and hypogonadism (in adults) being the most frequently reported. One or more pituitary hormone deficits may be already present at the time of craniopharyngioma detection in a significant number of patients necessitating early diagnosis and appropriate treatment.

Renewed interest in the use of therapeutic enzymes combined with an improved knowledge of cancer cell metabolism, has led to the translation of several arginine depletion strategies into early phase clinical trials. Arginine auxotrophic tumors are reliant on extracellular arginine, due to the downregulation of arginosuccinate synthetase or ornithine transcarbamylase-key enzymes for intracellular arginine recycling. Engineered arginine catabolic enzymes such as recombinant human arginase (rh-Arg1-PEG) and arginine deiminase (ADI-PEG) have demonstrated cytotoxicity against arginine auxotrophic tumors. In this review, we discuss the molecular events triggered by extracellular arginine depletion that contribute to tumor cell death.

Acute Lymphoblastic Leukaemia (ALL) is the most common leukaemia of childhood. Despite improvements in overall survival, significant numbers of children die from relapsed/ refractory disease or suffer the morbidities of multi-agent chemotherapy regimens. A major challenge is to find new drugs which are active against ALL blasts or which make blasts more sensitive to existing chemotherapy, without adding to toxicity. Arginine, is a semi-essential amino acid, required for protein synthesis, and cell viability. Some cancers are dependent on arginine for proliferation and survival - 'arginine auxotrophism'. We have recently identified that arginine metabolism plays a key role in AML pathogenesis and can be therapeutically targeted. Therefore we investigated if Acute Lymphoblastic Leukaemia is also sensitive to therapeutic arginine depletion. A clinical grade, FDA approved, pegylated-recombinant human arginase, PEG-rhArgI molecule BCT-100, has been developed by Bio-Cancer Treatment International (BCT, Hong Kong).A Phase I and II clinical trial has been successfully completed in adult hepatocellular carcinoma. The Phase I trial demonstrated that at 1600U/kg BCT-100 (Observed Biological Dose), plasma arginine falls below 8mM (Adequate Arginine Depletion - ADD) and is maintained for up to 166 hours, with no evidence for neutralising immunogenicity. Only grade 1 or 2 reversible transaminitis was seen and patients experienced significant improvements in overall survival. Arginine is recycled by cells from ornithine, through the enzymes OTC and ASS. These enzymes have been identified as biomarkers of sensitivity to arginine depletion in solid tumours. ASS and OTC expression pattern was characterised in leukaemic blasts from 37 ALL patients (19 adult, 18 paediatric), revealing the majority of ALL cases are deficient in one or more of these enzymes (Adults: ASS-: 84% OTC: 53%; Paediatric: ASS- 44% OTC 27%). Thus the majority of ALL blasts are auxotrophic for arginine. We tested the cytotoxicity of BCT-100 against ALL. We demonstrated that ALL cell lines (n=4) are dependent on arginine for proliferation and survival. In vitro, BCT-100 leads to a complete depletion of extracellular arginine and induction of autophagy. BCT-100 was tested against primary blasts from ALL patients - IC50S ranged from 17.5-2400ng/ml. Sensitivity to BCT-100 did not correlate with clinical or cytogenetic characteristics. The study is the first report on the arginine dependence of adult and paediatric ALL and provides the rationale for ongoing translation of BCT-100 for patients with relapsed ALL. Disclosures No relevant conflicts of interest to declare.