Tumour arginine addiction subverts the anti-cancer immune response - PowerPoint PPT Presentation

Tumour arginine addiction subverts the anti-cancer immune response Francis Mussai University of Birmingham

Overview • Acute Myeloid Leukaemia (AML) – The second most common leukaemia of childhood and increasingly common with older age • Neuroblastoma – The most common extra-cranial solid tumour of childhood • Common themes Common themes – Embryonic cells with developmental arrest and failure to mature, in combination with malignant proliferation – Immune alterations – Cytopenias at diagnosis (not due to bone marrow effacement) – Poor prognosis

Whole body Arginine Homeostasis L-arginine is an amino acid found in virtually all peptides, enzymes and proteins. • • Key metabolic pathways and cellular events – Urea Cycle Adrenal Adrenal Adrenal Cortex Cortex Cortex Liver Liver Liver CITRULLINE CITRULLINE CITRULLINE (ASS/ ASL) (ASS/ ASL) (ASS/ ASL) – Nitric Oxide formation Urea Cycle Urea Cycle Urea Cycle ARGININE ARGININE ARGININE – Creatine and polyamine production Cell cycle signaling – Ammonia Ammonia Ammonia – Protein synthesis ARGININE ARGININE ARGININE Kidneys Kidneys Kidneys Glutaminase Glutaminase Glutaminase Glutamate Glutamate Glutamate Glutamate Glutamate Glutamate • Consumed in the diet (85%) Small Intestines Small Intestines Small Intestines • Arginine can also be synthetised (5-15%) (simple view): – Glutamine converted to citrulline and ammonia in the enterocytes (Ammonia enters the urea cycle in the liver) – – Citrulline passes through the liver and converted to arginine in the proximal tubules of kidney via Argininosuccinate Synthetase/ Argininosuccinate Lyase (ASS/ASL) enzyme system. Arginine is released back into the blood – Added complexity (liver produced arginine is metabolised by the liver; dietary changes) • Tight compartmentalization in various organs and tissue spaces and under stringent homeostatic control in blood (NR:70-120 uM).

Intracellular Arginine homeostasis • Uptake via CAT family of surface proteins – ? A mechanism of arginine sensing • The majority of somatic cells can use citrulline as a surrogate to resynthesise arginine via resynthesise arginine via intracellular ASS/ASL/OTC • Arginine is considered a semi- essential amino acid – under conditions of high demand (pregnancy, inflammation, trauma) whole body arginine can become limiting

Myeloid cells and arginine • Non-malignant myeloid cells metabolise arginine to alter the balance of immunity – Tumour-associated – Tumour-associated Macrophages (TAMs) Macrophages (TAMs) – Myeloid-derived suppressor cells (MDSCs) • Cancer ( Reviewed in Mussai et al. 2011) • Infection • Pregnancy Gabrilovich, Ostrand-Rosenberg, Bronte 2012

Myeloid-T interactions

Arginine addiction by cancer cells • Certain cancers are unable to re-synthesise arginine because of Hepatocellular carcinoma (Cheng 2007) Prostate cancer (Hsueh 2012) down-regulated, or Pancreatic cancer (Glazer 2011) Renal cell carcinoma (Yoon 2007) absent expression of OTC/ absent expression of OTC/ Breast cancer (Wang 2014, Qiu 2014) Breast cancer (Wang 2014, Qiu 2014) Lung/Mesothelioma(Agrawal2012; Szlosarek 2006) ASS/ ASL Melanomas (Lam 2011; Yoon 2013) Glioblastomas (Sippel 2011) (Pavlyk 2015) • Malignant cell is reliant Diffuse Intrinsic Pontine Glioma (Ching, Baylor) Osteosarcoma and lymphoma(Wells 2013; Kobayashi 2010) on extracellular arginine – Non-Hodgkin’s Lymphoma (Zeng 2013; Shu 2014) auxotrophism Neuroblastoma (Mussai et al. 2015; Lin et al. 2014) Acute Myeloid Leukaemia (Mussai 2015) • In these cases arginine is Acute Lymphoblastic Leukaemia (Mussai 2015) T-Acute Lymphoblastic Leukaemia (Kwong-Lam 2013) therefore an essential Soft tissue sarcoma (Yan 2011, Takaku 1995) amino acid

Observations in AML Observations in AML

Impaired anti-AML T cell immunity Post-therapy (TP:3) Diagnosis (TP:1) SSC SSC SSC SSC 33 17 FSC FSC FSC CD8 CD8 Cancer Testis Antigen responses =TP:1 (diagnosis) =TP:2 (cycle 3) =TP:3 (cycle 6)

AML blasts express classical MDSC phenotype CD14+ or CD15+ - dependent on maturation stage

AML blasts consume extracellular arginine Plasma arginine Arginine transport Arginine catabolism

Arginine ASS/ASL ARG Citrulline Ornithine OTC

AML blasts are auxotrophic for arginine Adult Paediatric

Dependence on extracellular arginine for survival For more on this story see Mussai et al. Blood 2015

MDSCs suppress T cell immunity by arginine depletion AML blasts ARGININE ? DEPLETION

AML blasts suppress T cell proliferation T cells DC :AML blast:Tcells MLR =Mixed Leukocyte Reaction 5x10 4 : ?x10 5 : 2x10 5 DC 100 %) P1 P1 P9 P9 T cell proliferation (% P2 P10 80 P3 P11 60 P4 P12 P5 P13 40 P6 P14 P7 P15 20 P8 0 1:1 1:0.5 1:0.25 1:0.125 1:0 T cells:AML blasts Mussai et al. Blood 2013

Arginase inhibitors restore T cell proliferation

Arginine metabolism in AML Low/absent expression of ARG1 and iNOS in AML….what’s going on?

Arginase II in AML blasts Secondary alone ARGINASE II ELISA

Arginase I vs II • ARG I – Chromosome 6 – Hepatocytes (Morris et al. 2007) – Cytoplasmic KO in mice is lethal (Iyer et al. Mol Cell Biol 2002) – – Deficiency in humuans leads to profound hyperargininemia, neurodisability/toxicity, organ dysfunction (Schlune et al. Amino Acids 2015) organ dysfunction (Schlune et al. Amino Acids 2015) • ARG II – Chromosome 14 (?gene duplication) – 60% amino acid sequence homology. 354 aa (Colleluori et al. 2001) – Tissue localised – renal, neural, endothelial (Jenkinson et al. 1996) – Mitochondrial (imported via a 22-residue N terminal sequence) (Morris et al. 1996) – KO in mice is unsymptomatic (mild hyperargininemia) (Shi et al. 2001) – No human phenotype identified • Both convert arginine into urea and ornithine • Not much known about the role of Arginase II in immunity

AML extends the immunosuppressive microenvironment. ARGII release into the blood *** 15 ) 1500 a e e II (ng/ml) *** r U e l o m 10 1000 µ Plasma Arginase ( ( y t i v i t c a 5 500 e s a n i g r A 0 0 AML patients Healthy controls A M L p a t i e n t s e a t h y c o n t r o l s H l

T cell inhibition by the blood of AML patients T cells DC :Plasma:Tcells MLR =Mixed Leukocyte Reaction 5x10 4 : 50 µ l : 2x10 5 DC 150 100 *** T cell proliferation (%) plasma plasma+ arginine T cell proliferation (%) 80 plasma+ Inhibitors 100 60 40 50 20 0 0 Patient 7 Patient 15 Patient 4 Patient 1 Patient 12 AML patients Healthy controls

Summary 1 • AML is reliant on extracellular arginine and is unable to recycle arginine from precursors • ARGII expression depletes arginine in the local and systemic microenvironment through and systemic microenvironment through expression and release of ARGII • AML mimics MDSC phenotype Is this unique to AML?

Neuroblastoma tumour cells have arginase activity Cell lines TH-MycN mice Patients Mussai et al. Can Res 2015

Neuroblastoma expresses Arginase II Cell lines TH-MycN mice Patients Arginase II Anti rabbit ab

Resulting T cell suppression

Tumour mass lowers plasma arginine Patients TH-MycN mice Stage III/IV Large tumours

Impact on immunity Antigen specific immunity is impaired NY-ESO is the most prevalent cancer-testis antigen in NB

Impact on immunity CAR-T cell function is impaired Anti-GD2 CAR-T cells - new approach for NB Outcomes limited by failure of sustained CAR-T cell proliferation in patients

Neuroblastoma ARGII expression impacts survival

Summary 2 • Neuroblastoma expresses ARG2 and depletes local and systemic arginine – (Neuroblastoma is auxotrophic for arginine) • Modulation of – Haematopoiesis – Haematopoiesis – Myeloid cell immunity – T cell immunity • Autologous • Antigen-specific • Engineered CAR T cells • Associated with a worse prognosis

Summary 3 • Tumours import arginine for survival and proliferation • Lowers local and systemic levels of arginine • Modulates surrounding myeloid cells to an • Modulates surrounding myeloid cells to an immunosuppressive phenotype • Inhibits autologous T cell proliferation • Suppresses engineered therapeutic T cell approaches

Thank you Great Ormond Street University of Birmingham • John Anderson • Carmela De Santo • Jon Fisher • Joseph Higginbotham-Jones Pam Kearns • • Tracey Perry Oxford University • Charlie Craddock, AML Working Party • Kate Wheeler • • Justin Loke and Guy Pratt Justin Loke and Guy Pratt • Jennie Godwin University of Nottingham • Sharon Egan Staff, patients and parents Institute of Cancer Research • Lou Chesler Hannah Webber • • Laura Danielson Bio-Cancer Treatment International • Paul Cheng