This is a repository copy of Ionic modulation of immune checkpoint proteins.

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Conference or Workshop Item: Shandell, Mia Ann orcid.org/0000-0002-7864-758X, Lawrence, Samantha, Brackenbury, Will orcid.org/0000-0001-6882-3351 et al. (1 more author) (2019) Ionic modulation of immune checkpoint proteins. In: Physiology 2019, 07-10 Jul 2019, Aberdeen Conference Centre.

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[email protected] https://eprints.whiterose.ac.uk/ Ionic modulation of immune checkpoint proteins Mia A. Shandell1,2,3, Samantha M. Lawrence1,2 , William J. Brackenbury1,2 , Dimitris Lagos 1,3 1. York Biomedical Research Institute. University of York. York, UK. 2. Department of Biology. University of York. York, UK. 3. Hull York Medical School. University of York. York, UK.

Abstract Results Despite extensive basic and clinical research on immune TNF-α/IFN-γ stimulates IDO1 expression in lung cancer A549 and breast checkpoint regulatory pathways, little is known about the cancer MDA-MB-231 cells. effects of the ionic tumour microenvironment on immune MDA-MB-231 checkpoint expression and function. A) A549 B) IDO1 activity C) Kynurenine standard curve A549 0.8

80 ) kDa αγ αγ untreated y = 0.004428x + 0.2388

UT UT m

n 2 ) TNF-a/IFN-g R = 0.9982 2 M 60 0.6

55 9 m ( 4

MDA-MB-231 ( e

e IDO1 n

We screened effects of modulating i

40 untreated c n 0.4 n

35 e r TNF-a/IFN-g a u b r compounds on IDO1 activity. Here, we describe a n o y 20 k s 0.2

+ + b

35 GAPDH A mechanistic link between Na /K ATPase inhibition by 0 0.0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 20 40 60 80 100 0 0 0 0 0 0 0 0 0 0 0 1 2 3 4 5 6 7 8 9 0 1 cardiac glycosides and activity of indoleamine-2',3'- 1 1 m cell number kynurenine ( M)

dioxygenase (IDO1), a well-characterized immune A) Western blot of IDO1 in A549 and MDA-MB-231 cells +/- 25 ng/mL TNF-α/1000 U/mL IFN-γ stimulation. B) IDO1 assay cell titration for A549 and MDA-MB-231 cells. Used 10,000 A549/well and 50,000 MDA-MB-231/well as seeding density in all following experiments. C) Representative kynurenine absorbance standard checkpoint. curve calculated from kynurenine standards in 0.5M HCl. Cardiac glycosides ouabain and digoxin inhibit kynurenine production IDO1 catalyses the rate-limiting step of tryptophan in A549 and MDA-MB-231 cancer cells with a modest impact on cell survival. catabolism and inhibits the immune response to the A) B) C) tumour by local depletion of tryptophan, an amino acid A549 - IDO1 assay A549 alamar blue t = 4h A549 n ) o l i 0.6 2.0 t 150 ouabain

o ouabain a ouabain r t v

essential for anabolic functions in cancer and T cells. i digoxin

n digoxin t o c digoxin 0 0 a c 6 / 100 1.5 r s 1 0.4 o b O t i A D

b I - i

f 0

h 1.0 7 o 50

n 5

Methods i s n (

b o y i 0.2 t t A i i

l 0.5 i b i

0 b h a i n

IDO1 activity is measured using a kynurenine assay. i

V

% -4 -2 0 2 0.0 0.0 10 10 10 10 10 -5 10-4 10 -3 10 -2 10 -1 0.0 0.5 1.0 1.5 m Day 1 Alamar Blue assay log(inhibitor) ( M) log(inhibitor) (mM) IDO1 activity (inhibitor/control) Seed cells Treat with compound (after removing supernatant in step (1) Add 10 uL Alamar Blue to cells in tissue culture plate. Incubate 4h 37C/5% CO2. Measure absorbance at 570 nm, 600 nm. D) E) F) MDA-MB-231 alamar blue t = 4h MDA-MB-231 - IDO1 assay MDA-MB-231

Day 2 n o 0.6 ) i Kynurenine assay of IDO1 activity l Stimulate with 25 ng/mL TNF-α t 1.5 ouabain o a ouabain ouabain r t v 100 digoxin and 1000 U/mL IFN-γ (1) Transfer 150 μL supernatant to a (2) Centrifuge 10 min/3000 rpm/room temperature. i n t digoxin 0 c

digoxin o

round-bottom plate. Transfer 100 μL to new plate. 0 a c 6

80 / s r +10 μL 30% v/v trichloroacetic acid. (3) +100 μL 1.2% w/v Ehrlich’s reagent 1 0.4 b Day 3 o 1.0 O 50°C/30 min in acetic acid. Room temperature/10 min t i A D

b I Kynurenine, Alamar Blue assays 60 -

(4) Measure absorbance at 492 nm. i

f 0 h 7 O o

n 5

Cl i n ( 40 s

o HN Cl OH b i O N 0.2 y 0.5 t N t A i O i

O Cl l b i

O + H2N N i 20

O b

HN h H2N H2N NH2 H2N a i IDO acetic acid n 30% v/v H2O CO2H i H

H V CO2H CO2H CO2H 10 min RT 0 50C/30 min % 0.0 10-1.5 10 -1.0 10 -0.5 10 0.0 0.0 -2.0 -1.5 -1.0 -0.5 0.0 0.0 0.5 1.0 1.5 tryptophan N-formylkynurenine kynurenine Ehrlich's reagent Absorbance: 492 nm 10 10 10 10 10 log(inhibitor) (mM) IDO1 activity (inhibitor/control) G) log(inhibitor) (mM) Ion channel drug screen of IDO1 activity IDO1 activity IC50 A549 MDA-MB-231

Drug/toxin Target(s) Effect )

l A549 + 4-aminopyridine Kv K channels Inhibitor o r ASIC channel Inhibitor t 150

n ouabain 17 nM 89 nM + 2+ Benzamil Na /Ca exchanger, ENaC, TRPP3, TRPA1 channels Inhibitor o c

Bupivacaine Nav, K2P channels Inhibitor + O * Carvedilol Kv1.5, Kir2.3 K channels, adrenoceptors Inhibitor S digoxin 40 nM ~164 nM

2+ M 100 Celecoxib L-type Ca and Kv7 channels both D

Diclofenac (sodium) Kv7 channels Activator % (

Eslicarbazepine acetate VGSC Inhibitor

2+ y dihydrochloride L-type and T-type Ca channels Inhibitor t **** i 50 A) Dose-response curves for ouabain and digoxin treatment of TNF-α/IFN-γ stimulated A549 cells. B) Alamar Blue survival assay after 4h incubation with reagent. v

Fluoxetine hydrochloride Kv4.3 channels Inhibitor i ****

t C) Comparison of IDO1 activity vs. viability as measured by Alamar Blue in A549 cells. D) Dose-response curves for ouabain and digoxin treatment of TNF-α/IFN-γ + c **** Iberiotoxin KCa1.1 K channels Inhibitor a stimulated MDA-MB-231 cells. E) Alamar Blue survival assay after 4h incubation with reagent. F) Comparison of IDO1 activity vs. viability as measured by Alamar Memantine hydrochloride Inhibitor

NMDA receptor 1 2+ Blue in MDA-MB-231 cells. G) Calculated IDO1 activity IC50s from fitting the dose-response curves by non-linear least squares regression. L-type Ca channels Inhibitor O 0 ) e il e l x c e e e n n e x n n e e D P b n e e i e x i e e 9 i i A 4 X + O S id m in ilo i ci n d T a IO n in in t d T il d in T n 1 a o in 3 in T I S A x i i i i i i E - NS-1619 K 1.1 K channels Activator B - r a a d o a lo r h n B p iz t n Ib ic t g p 6 b yt z T id T Ca M P 4 lo z c e c s o o C fe E e r e e m m n M i -1 a la M r D ( i n a v e p x ip o - z a x p la M ra a d u n o M A t M M m e iv r l o r M l 1 a n o a c M m e m M fe S o e n ra m + + O m a b p a e ca n a m ic b lu lu b n m p e m i N h a m R e % S u c c e c d M r f f a e 1 1 n M p r 0 T v 0 Ouabain Inhibitor 1 0 M M b b .1 m a g b . lo m 5 M m 3 1 Na /K ATPase M 0 m m M M M r M 0 M ic M M li 0 0 M m M M M M D 1 m m m a m m 0 l m m M g M M 0 m 0 m m m m 0 0 M 5 0 0 c 5 s 0 m m m 0 0 1 5 0 5 % 5 1 m 1 1 5 0 e 0 1 M . 0 . 0 A549 MDA-MB-231 VGSC, HERG Inhibitor 5 0 M .0 0 1 2 m 1 1 0 1 5 1 0 1 0 0 m 0 1 M . 1 m 0 + 0 3 0 1 Tetraethyl-ammonium chloride Inhibitor 0 K channels - all 1 VGSC Inhibitor *p<0.05 ANOVA, Bonferroni multiple comparisons + TRAM-34 KCa3.1 K channels Inhibitor Veratridine VGSC Activator

1-EBIO KCa3.1, KCa2 channels Activator

) MDA-MB-231 Capsaicin TRPV1 channels Inhibitor l Ouabain, but not digoxin, Carbenoxolone Pannexin-1 (Gap junction channel) Inhibitor o r Cariporide NHE1 Inhibitor t 150 kDa n + KCa1.1, Kv1.2, Kv1.3 K channels Inhibitor o c downregulates IDO1 expression in VGCC / alpha-2-delta Inhibitor 55 0.05%25 DMSO nM25 ouabain nM digoxin0.05%150 DMSO nM150 ouabain nM digoxin TRPA1, KATP channels Inhibitor O S Icilin TRPM8 channel Activator 100 * M ** 2+ A549 and MDA-MB-231 cancer cells. hydrochloride L-type, T-type Ca , HCN channels, NMDA, μ-opioid receptorsInhibitor D IDO1

Margatoxin Inhibitor Kv1.3, Kv1.6 % ( VGSC Inhibitor 35 y t i 50 v

i **** t 35 GAPDH A library of 31 compounds targeting ion channels or c

a ****

pumps was screened in the kynurenine assay measuring IDO1 1 O 0 activity in TNF-α/IFN-γ stimulated lung cancer (A549) and breast D il l e O ) P e e o ib in e e x c O e e e in e x in e e x e 9 in in A 4 e X I S d n l n d T a I n n n t d l d n T n 1 n 3 n S -A ri m i i x ic o ri h n i zi ti n i T i i ti g i a to zi E - i T B o a a d o a l e B p i e e m Ib ic m n ip 6 b y T id T M P 4 il z c e c s o o C f E e r x p a a M d -1 a n la M tr TNF-α/IFN-γ ( n a v e x ip o - z a l M a u o A M D M m e iv r l p o r M l 1 a n o a c M r m fe S o e n M ra cancer (MDA-MB-231) cells. Points (right panel) represent O m a b a e a n a m ic b lu lu b n m m e e M i N h a m R e m % S p c c c e c d M r f f a e 1 1 p m n M p r 0 T v 0 1 0 M M u b 1 m a g b . lo m 5 M 3 1 M 0 m b M M M r M . M ic M M li 0 0 M m m M M M M D 1 m m m m a m 0 m 0 l m m M g M M 0 m 0 m m m 0 0 M 5 0 c 5 s m m m 0 1 5 m technical replicates and data are normalized to their respective % 5 1 m 0 1 5 0 e 0 0 M . 0 0 0 .5 0 5 0 1 M .0 0 1 1 2 m 1 1 0 1 5 1 0 1 Western blot of IDO1 expression in TNF-α/IFN-γ stimulated cells treated with cardiac glycosides. A549 cells were treated with 0.05% DMSO, 25 nM ouabain, 0 0 m 0 1 M . 1 m 0 0 3 0 1 0 DMSO control, 0.05% or 1% DMSO in PBS. 1 or 25 nM digoxin (left panel). MDA-MB-231 cells were treated with 0.05% DMSO, 150 nM ouabain, or 150 nM digoxin (right panel). *p<0.05 ANOVA, Bonferroni multiple comparisons Cardiac glycosides decrease activity of immune checkpoint protein IDO1 in cancer cells.