TAT 2015 PARIS

2015-03-03

MTH1 inhibitors in cancer treatment Thomas Helleday

Disclosures: Inventor MTH1 inhibitor patents

Personlized cancer treatment (genotype) – the future? o Extensive genetic buffering and cancers can change genotype. o Intra tumour heterogeneity – is the biopsy representative? o Can we predict phenotype from genotype? Microenvironment? o Are current targeted therapies curing cancer?

Ideal treatment of cancer o Should work on heterogeneous cancers o Should work for all patients o Should be curative

CHARACTERISTICS OF AN ANTI-CANCER TARGET?  Cause DNA damage to kill cancer cells  Exploit the high level of endogenous DNA damage in cancer

 Target replicating cells  Target non-essential enzyme that is required for survival in cancer cells  Druggable

MutT homologue - MTH1

MTH1 sanitise oxidated dNTPs to prevent DNA damage 2-OH-dAMP 8-oxo-dGMP

2-OH-dATP 8-oxo-dGTP

MTH1

dNTP

DNA damage MTH1 inhibition

 Bases in free dNTPs are damaged 102–105 fold more effectively than in DNA (Topal and Baker 1982)  MTH1 overexpression reverts mutator phenotype in mismatch repair defective cells (Russo et al. 2004)  MTH1 overexpression inhibits RAS-induced DNA damage and premature senescence (Rai et al. 2011)  MTH1 levels and activity is increased in cancer (Speina et al. 2005; Obtułowicz et al. 2010)

Catalytic activity of MTH1 required for survival

Tubulin

VH10

U2OS E56A

MTH1

Gad, 2014 Nature 508:215-21

MTH1 siRNA

N M N M N M

NT siRNA

E56A

+DNA

U2OS

wt

U2OS WT

-

U2OS

8-oxodG

53BP1 foci, ATM activation, repair and apoptosis

+WT +E56A

MTH1 siRNA

control

NT siRNA

53BP1

ATM pS1981

DNA

RAD51

DNA

DNAPKcs pS2056

DNA

Cleaved Caspase 3

MTH1 is required for tumour growth NT RNA

dox

-

+

MTH1 shRNA

-

+

tubulin

MTH1

Survival (% of uninduced)

120 100 80 60 40 20 0 NT RNA MTH1 sh#2

Gad, 2014 Nature 508:215-21

MTH1 AS AN ANTI-CANCER TARGET?  Cause DNA damage to kill cancer cells   Exploit the high level of endogenous DNA damage in cancer   Target replicating cells   Target non-essential enzyme that is required for survival in cancer cells   Druggable

Screen for MTH1 inhibitor o Several screening campaigns

o Good permeability

o Hits; molecular weight < 500

o Solubility (range of soluble to less soluble)

o Solved MTH1 crystal structure

o Human metabolic stability o Optimising for mouse metabolic stability

(Svensson et al 2011) o Several co-crystals, supporting SAR and pharmacophore

IC50 = 3 µM

IC50 = 3.6 nM

IC50 = 0.8 nM

TH086

TH287

Human Metabolic Stability

TH588 binds MTH1 protein

5

111 nM 33 nM

re s p o n s e (R U )

4

11 nM 3 ,3 n M 1 ,1 n M

3 2 1 0 100 -1

Gad, 2014 Nature 508:215-21

200

t im e ( s e c o n d s )

300

TH588 increases incorporation of 8-oxodGTP into DNA in cancer but not primary cells *

U2OS

*

70

Tail moment

60

50 40 30 20

10 0 control Ogg1 control Ogg1 control Ogg1 DMSO

TH588

TH287

70

VH10

Tail moment

60 50 40 30 20 10 0

control Ogg1 control Ogg1 control Ogg1 DMSO

Gad, 2014 Nature 508:215-21

TH588

TH287

MTH1 inhibitors kill cancer cells

10%

1%

Survival (% control)

VH10 HDFn HAEB U2OS HeLa MDA-MB-231 MCF-7 SW480 SW620

140

hTERT

120

RasV12

100

SV40T

80 60 40 20

2

3

4

5

6

7

8

9

10 0

[TH588] (µM) 300

0

10 20 [TH588] (µM) Concentration (µM)

** *

250 200

ns

150 100 50 0

U2OS

TH287 TH588 TH650

1

TH287 TH588 TH650

0

8-oxo-dG

Clonogenic survival

100%

VH10

TH588 IC50 = 4 nM

MTH1 inhibitors cause 53BP1, RPA and DNA-PKcs foci

20 15 TH287 10

TH588

5

TH650

RPA pos. cells (%)

0

25 20 15

TH287

10

TH588

5

TH650

0 0

2

4

6

8

10

0

TH287 10

TH588

5

TH650

0 6

8

10

Concentration (µM)

Gad, 2014 Nature 508:215-21

RPA pos. cells (%)

53BP1 pos. cells (%)

15

4

2

4

6

8

TH588

5

10

TH650 0

2

4

6

8

10

Concentration (µM) 20

VH10

30

20

2

TH287

10

Concentration (µM)

VH10

0

15

0

Concentration (µM) 25

U2OS

20

25 20 15

TH287

10

TH588

5

TH650

DNA-PKcs pos. cells (%)

53BP1 pos. cells (%)

U2OS

30

DNA-PKcs pos. cells (%)

U2OS

25

VH10

15 TH287

10

TH588

5

TH650

0

0 0

2

4

6

8

Concentration (µM)

10

0

2

4

6

8

Concentration (µM)

10

Caspase 3 pos . cells (%)

MTH1 inhibitors trigger DDR and apoptosis 20 15 10

TH588 TH287

5 0 0

2

4

6

8

10

Concentration (µM)

Sub-G1 pso. cells (%)

25 20 15 TH588

10

TH287

5 0 0

2

4

6

8

Concentration (%) ATM pS1981

Gad, 2014 Nature 508:215-21

10

Once daily treatment show response in colorectal and breast cancer xenografts (30 mg/kg s.c. q.d.)

(30 mg/kg s.c. q.d.)

TH588 has anti-tumour effects in patient derived xenograft

800 vehicle TH588

BRAF V600E mutated tumour from a malignant melanoma patient that developed resistance to Carboplatin/dacarbazine and vemurafenib

% Tumour growth

700 600 500 400 300 200 100 0 0

10 20 Days of treatment

Selectivity of MTH1 inhibitors?

TH287 and TH588 are selective MTH1 inhibitors

Gad, 2014 Nature 508:215-21

TH588 is a selective MTH1 inhibitor Assay 5-HT transporter 5-HT1A 5-HT1B 5-HT2A 5-HT2B 5-HT2C 5-HT3 5-HT5a 5-HT6 5-HT7 A1 A2A A3 alpha 1 alpha 2 AT1 AT2 B2 BB beta 1 beta 2 BZD BZD (central) Ca2+ channel CB1 CCK1 (CCKA) CCK2 (CCKB) CCR1 CDC2/CDK1 (cycB) CDK2 (cycA) CGRP Cl- channel CXCR2 (IL-8B) D1 D2S D3 D4.4 D5 delta 2 (DOP) dopamine transp EP2 EP4 ETA

% Inhibition 7 18 14 42 92 22 0 33 15 40 79 81 48 27 57 5 12 1 -1 -7 2 44 -37 29 3 34 -6 -6 31 31 -1 66 -1 44 17 28 12 47 13 39 22 10 -16

Assay % Inhibition ETB -11 GABA 0 GAL1 -18 GAL2 -4 GR 9 H1 -10 H2 2 hERG 19 IP (PGI2) 18 kappa 38 KV channel -2 M1 7 M2 24 M3 7 M4 18 M5 -1 MC4 3 MT1 59 mTOR kinase 20 mu (MOP) 13 Na+ channel 34 NK1 63 NK2 6 NK3 51 NOP (ORL1) 4 norepinephrine transp. 99 NTS1 (NT1) -12 P2X 1 P2Y 58 PAC1 (PACAP) -10 PCP -2 PDGF -51 PDK1 -4 PPARgamma 27 sigma 40 SKCa channel -2 Src kinase 6 sst -13 TNF-alpha 0 V1a 22 VPAC1 (VIP1) 2 Y1 -12 Y2 -8

Norepinephrine transporter (NET) is a monoamine transporter and is responsible for reuptake of extracellular noradrenaline. NET is a targets of many antidepressants and recreational drugs

% Inhibition at 10 µM 95%

Expression of E. coli MutT rescues toxicity of TH588

45 40 Viability (%)

35 30 25 20 15 10 5 0

53BP1 pos cells (%)

control MutT-m MutT-mn

10

control

8

MutT-m

6 4 2 0 0

2

4 6 8 [TH588] µM

10

Cancer genotype

Targeting oncogenes normal

cancer mutation

Targets activating mutation HER-2, B-RAF, EGFR, BCR-ABL, EML4-ALK

Cancer phenotype

Targeting tumour suppressors normal

cancer

Targeting dependencies normal

cancer

mutation

Non-essential target becoming essential by other gene mutation

Non-essential target becoming essential in cancer

PARP

MTH1

synthetic lethality

cancer phenotypic lethality

open innovation

TAKE HOME MESSAGE o MTH1 inhibitors effective against many cancers o Targets a non-essential enzyme (mild side effects)

o Orally available (pills) o Effective against multi-resistant cancers o Can potentially be combined with current chemotherapy or targeted therapy.

Acknowledgements Biology Helge Gad Saeed Eshtad Cecilia E. Ström Fredrik Johansson Lars Bräutigam Andreas Höglund Anna Hagenkort Mikael Altun Bastiaan Evers Tatjana Djureinovic Jordi Carreras Puigvert Cecilia Lundin Kumar Sanjiv Niklas Schultz

Medicinal Chemistry Tobias Koolmeister Sylvain A. Jacques Marie-Caroline Jacques-Cordonnier Matthieu Desroses Evert J. Homan Karl S. A. Vallin Olov A. Wallner Martin Scobie

Biochemistry Olga Loseva Ann-Sofie Jemth

In vitro assay Elisee Wiita Ingrid Almlöf Christina Kaldéren Fredrik Jeppsson Kia Strömberg

In vivo Pharmacology Camilla Göktürk Pawel Baranczewski Therese Pham Fabienne Z. Gaugaz Ulrika Warpman Berglund

CBCS Anna-Lena Gustavsson Lars Johansson Martin Henriksson Lars G.J. Hammarström Annika Jenmalm Jensen Thomas Lundbäck Hanna Axelsson

Cell screening Bo Lundgren Maria Häggblad Ulf Martens

UDOPP (Uppsala) Stockholm University Linda M. Svensson Ronnie Berntsson Robert Gustafsson Pål Stenmark

Gothenburg University Jonas A. Nilsson Berglind Osk Einarsdottir Roger Olofsson

Ingrid Granelli Aljona Saleh

Linköping University Svante Vikingsson

Richard Svensson Per Artursson

Positions available

PSF Helena Berglund