Immune checkpoint blockade has resulted in long-lasting patient survival. However, majority of patients do not benefit from current immunotherapies because tumors employ an arsenal of evasion mechanisms. We used the iOTarg RNAi screening platform to discover targets and pathways in tumor cells that induce immune resistance. Here we report that desensitization to immune cell-derived TNF is a key immune evasion strategy of tumor cells, which is tightly regulated by the tumor-intrinsic activity of salt-inducible kinase 3 (SIK3). SIK3 potentiates NFkB activity in a TNF-rich environment, thereby negating its pro-apoptotic effect. Using small molecule inhibitors, we show that specific abrogation of SIK3 results in broad anti-tumor activity against multiple human tumor cell lines in vitro as well as in a significant tumor growth inhibition in MC38 syngeneic mouse model. Taken together, targeting SIK3 to re-sensitize tumors to immune attack is a compelling therapeutic strategy for cancer treatment.

→→ iOtarg platform identifies SIK3 as a novel tumor-associated immune checkpoint

→→ SIK3 desensitizes tumor cells to TNF-mediated apoptosis

→→ Intratumoral SIK3 activity strongly increases chromatin accessibility and expression of NFkB-regulated genes by preventing HDAC4 shuttling into the nucleus

→→ SIK3-specific inhibitors alone sensitize a wide array of human and murine cancer cell lines to TNF-induced apoptosis by engaging the HDAC4/NFkB pathway

→→ Our novel SIK3 inhibitor demonstrates significant anti-tumor effect in MC38 syngeneic mouse model combined with an improved anti-tumor immune profile

TCR

MHC

TNF

TNFR1

LKB1

SIK3

HDAC4

HDAC4

NFκB

NFκB

TRADD

TRAF2

IκB

IKKMEKK3

Deathcomplex

caspases

e�ectorcaspases

Apoptosis

Survival

AS1 Mode of Action

iOTarg identifies and validates SIK3 as an immune checkpoint in multiple solid tumors

→→ SIK3 outperformed PD-L1 in the pancreatic cancer iOTarg screen→→ Knockdown of SIK3 sensitized multiple other tumors to T cell-

mediated killing→→ Conversly, SIK3 overexpression abrogated TIL-mediated killing

arrayed siRNA library + / -

co-culture

Tumor cell line

Patient-derived TIL culture

Immunecheckpoint

Immune activator

Negativecontrol

Negativecontrol

PANC1 : TIL#1

siCtrl s1 s2 s3

siPD-L1

0

25

50

75

100

rem

aini

ng tu

mor

cell

viab

ility

[%]

********

****

****

siSIk3

M579 : TIL209

MelanomaM579 : TIL209

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Colorectal cancerSW480 : TIL#1

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Breast cancerMCF7 : survivin-TCs

siCtrl

siCtrl

siSIK3

siSIk3

TCs

+

-

Tum

or ce

ll dea

th (µ

m2 /w

ell)

0 15 300

5x105

1x106

0 20 400

5x106

1x107

Time (h)0 10 20 30 40 50 60 70

0

1x106

2x106

3x106

4x106

5x106

shCtrl

shCtrl

shSIK3

shSIK3

TCs

+

-

siCtrl

siCtrl

siSIK3

siSIK3

TCs

+

-

0

25

50

75

100

*

EV OE

rem

aini

ng tu

mor

cell

viab

ility

[%]

A iOtarg screening overview

siRNA validation

AS1 Target validation in solid cancers

E Overexpression of AS1 rescues tumor cells from TC-lysis

Pancreatic cancer screening results

−4

−2

0

2

4

6

0 500 1000 1500 2000 2500 3000Gene rank

TC-m

edia

ted

lysi

s (LO

ESS

scor

e)

siSIK3

siCEACAM6siPD-L1

siCtrl

Potential immunemodulators

D

B C

Ag-specific MIL cell culture

Tumor cell culture

Luciferase

Transfection

Co-culture

Lysis

Residual luciferase

Luci

fera

se U

nits

Luci

fera

se U

nits

Control si

RNA

Immune-checkpoint

siRNA

Control si

RNA

Immune-checkpoint

siRNA

Read-out

Cytotoxicity setup Viability setup

Screening

PANC1 : TIL#1

siCtrl s1 s2 s3

siPD-L1

0

25

50

75

100

rem

aini

ng tu

mor

cell

viab

ility

[%]

********

****

****

siSIk3

M579 : TIL209

MelanomaM579 : TIL209

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Colorectal cancerSW480 : TIL#1

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Breast cancerMCF7 : survivin-TCs

siCtrl

siCtrl

siSIK3

siSIk3

TCs

+

-

Tum

or ce

ll dea

th (µ

m2 /w

ell)

0 15 300

5x105

1x106

0 20 400

5x106

1x107

Time (h)0 10 20 30 40 50 60 70

0

1x106

2x106

3x106

4x106

5x106

shCtrl

shCtrl

shSIK3

shSIK3

TCs

+

-

siCtrl

siCtrl

siSIK3

siSIK3

TCs

+

-

0

25

50

75

100

*

EV OE

rem

aini

ng tu

mor

cell

viab

ility

[%]

A iOtarg screening overview

siRNA validation

AS1 Target validation in solid cancers

E Overexpression of AS1 rescues tumor cells from TC-lysis

Pancreatic cancer screening results

−4

−2

0

2

4

6

0 500 1000 1500 2000 2500 3000Gene rank

TC-m

edia

ted

lysi

s (LO

ESS

scor

e)

siSIK3

siCEACAM6siPD-L1

siCtrl

Potential immunemodulators

D

B C

Ag-specific MIL cell culture

Tumor cell culture

Luciferase

Transfection

Co-culture

Lysis

Residual luciferase

Luci

fera

se U

nits

Luci

fera

se U

nits

Control si

RNA

Immune-checkpoint

siRNA

Control si

RNA

Immune-checkpoint

siRNA

Read-out

Cytotoxicity setup Viability setup

Screening

PANC1 : TIL#1

siCtrl s1 s2 s3

siPD-L1

0

25

50

75

100

rem

aini

ng tu

mor

cell

viab

ility

[%]

********

****

****

siSIk3

M579 : TIL209

MelanomaM579 : TIL209

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Colorectal cancerSW480 : TIL#1

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Breast cancerMCF7 : survivin-TCs

siCtrl

siCtrl

siSIK3

siSIk3

TCs

+

-

Tum

or ce

ll dea

th (µ

m2 /w

ell)

0 15 300

5x105

1x106

0 20 400

5x106

1x107

Time (h)0 10 20 30 40 50 60 70

0

1x106

2x106

3x106

4x106

5x106

shCtrl

shCtrl

shSIK3

shSIK3

TCs

+

-

siCtrl

siCtrl

siSIK3

siSIK3

TCs

+

-

0

25

50

75

100

*

EV OE

rem

aini

ng tu

mor

cell

viab

ility

[%]

A iOtarg screening overview

siRNA validation

AS1 Target validation in solid cancers

E Overexpression of AS1 rescues tumor cells from TC-lysis

Pancreatic cancer screening results

−4

−2

0

2

4

6

0 500 1000 1500 2000 2500 3000Gene rank

TC-m

edia

ted

lysi

s (LO

ESS

scor

e)

siSIK3

siCEACAM6siPD-L1

siCtrl

Potential immunemodulators

D

B C

Ag-specific MIL cell culture

Tumor cell culture

Luciferase

Transfection

Co-culture

Lysis

Residual luciferase

Luci

fera

se U

nits

Luci

fera

se U

nits

Control si

RNA

Immune-checkpoint

siRNA

Control si

RNA

Immune-checkpoint

siRNA

Read-out

Cytotoxicity setup Viability setup

Screening

If not indicated otherwise, graphs are representative data from at least two independent experiments. Data points show mean +/- SEM. P-values were calculated using two-tailed student’s t-test * p < 0.05, ** p < 0.01, *** p < 0.01 and **** p < 0.0001.

Sundberg TB, Liang Y, Wu H, et al. Development of Chemical Probes for Investigation of Salt-Inducible Kinase Function in Vivo. ACS Chem Biol. 2016;11(8):2105–2111.

Khandelwal, N., Breinig, M., Speck, T., Michels, T., Kreutzer, C., Sorrentino, A., Sharma, A.K., Umansky, L., Conrad, H., Poschke, I., et al. (2015). A high-throughput RNAi screen for detection of immune-checkpoint molecules that mediate tumor resistance to cytotoxic T lymphocytes. EMBO molecular medicine 7, 450-463

Introduction

SIK3 Mode of Action

A iOtargTM screening overview

B Discovery of novel immune checkpoints in pancreatic cancer

E Role of SIK3 validated in multiple solid tumor entities

C Knockdown of SIK3 increases tumor lsyis

Conclusion

Results

Statistic information

References

Download

siSIK3

siCtrl100

80

60

40

20

0

120

Iso Abanti-TNF

Iso Abanti-TRAIL

Iso Abanti-FasL

Medium

+ activated TIL supernatant -

**

**

** ** ** **

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

Cell

viab

ility

-2 -1 0 1 2 3 40

1.4x107

8.0x106

1.6x107

1.2x107

1.0x107

6.0x106

4.0x106

2.0x106

siCtrl siSIK3

TNF[log(ng/mL)]

0 1 2 3 4 5 6Time (h)

Tum

or C

ell D

eath

(µm

2 /wel

l)

siCtrl

siCtrl

siAS1

siAS1

TNF

+

-

0

5x107

1x108

C

B

A

**

**

Sup. TILsCtrl Ab

anti-TNF

-

--

+

-+

+

+-

+

++-

+

+++-

120

100

80

40

20

0

140

60

siSIK3

D

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

TNFCtrl Ab

anti-TNFR1

-

--

+

--

+

-+

+

+-

0

100

80

40

20

60

*siSIK3

E

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

F

T cell Tumor

PD-1 PDL-1

TCR MHC

TRAIL/FasL

TNF

SIK3

Apoptosis

Intrinsic resistance

Extrinsic resistance

TRAILR/Fas

TNFR1

Schematic representation of extrinsic and intrinsic resistance to TC attack

Blockade of TNF rescues tumor cells from killing

AS1 knockdown mediated tumor lysis is TNF dependent

A Intrinsic versus extrinsic immune resistance

SIK3-specific inhibitors sensitize tumor cells to TNF by counteracting NFkB activity

→→ SIK3 tool inhibitor HG-9-91-01 strongly sensitized a variety of tumor cell lines towards TNF-mediated killing

→→ Mechanisticaly, the inhibitor counteracted TNF-induced NFkB activitation by inhibiting HDAC4 phosphorylation, which was not seen by a SIK3-inactive inhibitor

SIK3

/TNF

inde

x

−2

−1

0

●●●● ● ●●● ●● ●●● ●● ●● ●● ●● ●● ●●● ●● ●●●

● ●●● ●● ●● ●●●● ● ● ●●● ●● ● ●● ●● ● ●●● ● ●●● ● ● ●● ●● ●● ●● ●● ●● ●●● ● ●● ●●● ● ●●● ●●● ●● ●● ●●●●● ●●● ●●● ●● ●● ●● ● ●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ●● ●● ● ●●● ●● ●● ●●●● ●● ●●● ●● ● ●●●● ● ●

●

●

●●

●

●

●

●● ●

●

●●

3 5 7 9 11CD8A expression

[RSEM (log2)]

p < 0.01R2 = 0.42

SIK3

/TNF

inde

x

PRF1 / GZMA expression [RSEM (geometric mean, log2)]

−2

−1

0

●

●

●●

●

●

●

●●●

●

●●

●●● ● ● ●●● ●● ● ● ●● ● ●● ● ●● ●●● ● ●●●● ●● ●

● ●●● ●● ●●● ●●● ●● ● ●● ● ●●●● ●● ● ●●●● ●●●● ●●● ●●●● ●●● ● ●●●●● ●●● ●● ●● ●●● ●●● ●● ●● ● ●● ● ● ●●● ● ●● ●● ●● ●● ●●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ● ● ●●● ●●● ●● ●●●●●● ●● ●●● ●●● ●●●● ● ●

4 6 8

p < 0.01R2 = 0.48

++++++

+++++++++++++

+++++++++++++

++++++ +++++++++

+++++++ + +++++ + ++++ +

+

+

+ +

++ + + + + + + +

0

25

50

75

100

0 1 2 3 4 5 6 7 8

Follow−Up time [Years]

Surv

ival

pro

babi

lity

[%]

p = 0.0012

index low

index high

1 4 8 11 15 180

200

400

600

study days

***

Tum

or v

olum

e [m

m3 ] vehicle

OMX-0370 qd 100 mg/kg

OMX-0370 bid 100 mg/kg

p=0.06

Group treatment total daily dose[e.g. mg/k/d]

dosing route Cumulated

dosesmice

Analysis

1 vehicle 10 ml/kg QD x 18 p.o 18 10 FC/TG

2 OMX0370 100 mg /kg QD x 18 p.o 18 10 FC/TG

3 OMX0370 100 mg /kg BID x 18 p.o 36 10 FC/TG

Study start: tumor cell injection5x105 MC38 cells (s.c.)

Randomization: TV ~ 100 mm3Treatment start: p.o. 7q7d Last treatment

FACS analysisDay -10

Day 0Day 1 Day 18

vehicle

OMX370 qd

OMX370 bid0

2

4

6

CTL / T reg ratio

ratio

CTL

/ Tre

g

**

*

vehicle

OMX370 qd

OMX370 bid0

10

20

30

40

M2-like TAM(CD206+ MHC-II+)

% o

f CD4

5+ cells

*** ***

vehicle

OMX370 qd

OMX370 bid0

2

4

6

8

cytotoxic T cells(GrzB+)

*

**

% o

f CD4

5+ cells

vehicle

OMX370 qd

OMX370 bid0

2

4

6

cytotoxic T cells(CD25+ CD69+)

**

% o

f CD4

5+ cells

A In vivo e®icacy study design

B

Inhibition of promotes an anti-tumor immune phenotypeC

A SIK3-dependent gene expression pattern is associated with poor surviuval in PRADD

A In vivo efficacy study design

SIK3

/TNF

inde

x

−2

−1

0

●●●● ● ●●● ●● ●●● ●● ●● ●● ●● ●● ●●● ●● ●●●

● ●●● ●● ●● ●●●● ● ● ●●● ●● ● ●● ●● ● ●●● ● ●●● ● ● ●● ●● ●● ●● ●● ●● ●●● ● ●● ●●● ● ●●● ●●● ●● ●● ●●●●● ●●● ●●● ●● ●● ●● ● ●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ●● ●● ● ●●● ●● ●● ●●●● ●● ●●● ●● ● ●●●● ● ●

●

●

●●

●

●

●

●● ●

●

●●

3 5 7 9 11CD8A expression

[RSEM (log2)]

p < 0.01R2 = 0.42

SIK3

/TNF

inde

x

PRF1 / GZMA expression [RSEM (geometric mean, log2)]

−2

−1

0

●

●

●●

●

●

●

●●●

●

●●

●●● ● ● ●●● ●● ● ● ●● ● ●● ● ●● ●●● ● ●●●● ●● ●

● ●●● ●● ●●● ●●● ●● ● ●● ● ●●●● ●● ● ●●●● ●●●● ●●● ●●●● ●●● ● ●●●●● ●●● ●● ●● ●●● ●●● ●● ●● ● ●● ● ● ●●● ● ●● ●● ●● ●● ●●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ● ● ●●● ●●● ●● ●●●●●● ●● ●●● ●●● ●●●● ● ●

4 6 8

p < 0.01R2 = 0.48

++++++

+++++++++++++

+++++++++++++

++++++ +++++++++

+++++++ + +++++ + ++++ +

+

+

+ +

++ + + + + + + +

0

25

50

75

100

0 1 2 3 4 5 6 7 8

Follow−Up time [Years]

Surv

ival

pro

babi

lity

[%]

p = 0.0012

index low

index high

1 4 8 11 15 180

200

400

600

study days

***

Tum

or v

olum

e [m

m3 ] vehicle

OMX-0370 qd 100 mg/kg

OMX-0370 bid 100 mg/kg

p=0.06

Group treatment total daily dose[e.g. mg/k/d]

dosing route Cumulated

dosesmice

Analysis

1 vehicle 10 ml/kg QD x 18 p.o 18 10 FC/TG

2 OMX0370 100 mg /kg QD x 18 p.o 18 10 FC/TG

3 OMX0370 100 mg /kg BID x 18 p.o 36 10 FC/TG

Study start: tumor cell injection5x105 MC38 cells (s.c.)

Randomization: TV ~ 100 mm3Treatment start: p.o. 7q7d Last treatment

FACS analysisDay -10

Day 0Day 1 Day 18

vehicle

OMX370 qd

OMX370 bid0

2

4

6

CTL / T reg ratio

ratio

CTL

/ Tre

g

**

*

vehicle

OMX370 qd

OMX370 bid0

10

20

30

40

M2-like TAM(CD206+ MHC-II+)

% o

f CD4

5+ cells

*** ***

vehicle

OMX370 qd

OMX370 bid0

2

4

6

8

cytotoxic T cells(GrzB+)

*

**

% o

f CD4

5+ cells

vehicle

OMX370 qd

OMX370 bid0

2

4

6

cytotoxic T cells(CD25+ CD69+)

**

% o

f CD4

5+ cells

A In vivo e®icacy study design

B

Inhibition of promotes an anti-tumor immune phenotypeC

A SIK3-dependent gene expression pattern is associated with poor surviuval in PRADD

B Inhibition of SIK3 reduces tumor progression in vivo

SIK3

/TNF

inde

x

−2

−1

0

●●●● ● ●●● ●● ●●● ●● ●● ●● ●● ●● ●●● ●● ●●●

● ●●● ●● ●● ●●●● ● ● ●●● ●● ● ●● ●● ● ●●● ● ●●● ● ● ●● ●● ●● ●● ●● ●● ●●● ● ●● ●●● ● ●●● ●●● ●● ●● ●●●●● ●●● ●●● ●● ●● ●● ● ●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ●● ●● ● ●●● ●● ●● ●●●● ●● ●●● ●● ● ●●●● ● ●

●

●

●●

●

●

●

●● ●

●

●●

3 5 7 9 11CD8A expression

[RSEM (log2)]

p < 0.01R2 = 0.42

SIK3

/TNF

inde

x

PRF1 / GZMA expression [RSEM (geometric mean, log2)]

−2

−1

0

●

●

●●

●

●

●

●●●

●

●●

●●● ● ● ●●● ●● ● ● ●● ● ●● ● ●● ●●● ● ●●●● ●● ●

● ●●● ●● ●●● ●●● ●● ● ●● ● ●●●● ●● ● ●●●● ●●●● ●●● ●●●● ●●● ● ●●●●● ●●● ●● ●● ●●● ●●● ●● ●● ● ●● ● ● ●●● ● ●● ●● ●● ●● ●●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ● ● ●●● ●●● ●● ●●●●●● ●● ●●● ●●● ●●●● ● ●

4 6 8

p < 0.01R2 = 0.48

++++++

+++++++++++++

+++++++++++++

++++++ +++++++++

+++++++ + +++++ + ++++ +

+

+

+ +

++ + + + + + + +

0

25

50

75

100

0 1 2 3 4 5 6 7 8

Follow−Up time [Years]

Surv

ival

pro

babi

lity

[%]

p = 0.0012

index low

index high

1 4 8 11 15 180

200

400

600

study days

***

Tum

or v

olum

e [m

m3 ] vehicle

OMX-0370 qd 100 mg/kg

OMX-0370 bid 100 mg/kg

p=0.06

Group treatment total daily dose[e.g. mg/k/d]

dosing route Cumulated

dosesmice

Analysis

1 vehicle 10 ml/kg QD x 18 p.o 18 10 FC/TG

2 OMX0370 100 mg /kg QD x 18 p.o 18 10 FC/TG

3 OMX0370 100 mg /kg BID x 18 p.o 36 10 FC/TG

Study start: tumor cell injection5x105 MC38 cells (s.c.)

Randomization: TV ~ 100 mm3Treatment start: p.o. 7q7d Last treatment

FACS analysisDay -10

Day 0Day 1 Day 18

vehicle

OMX370 qd

OMX370 bid0

2

4

6

CTL / T reg ratio

ratio

CTL

/ Tre

g

**

*

vehicle

OMX370 qd

OMX370 bid0

10

20

30

40

M2-like TAM(CD206+ MHC-II+)

% o

f CD4

5+ cells

*** ***

vehicle

OMX370 qd

OMX370 bid0

2

4

6

8

cytotoxic T cells(GrzB+)

*

**

% o

f CD4

5+ cells

vehicle

OMX370 qd

OMX370 bid0

2

4

6

cytotoxic T cells(CD25+ CD69+)

**

% o

f CD4

5+ cells

A In vivo e®icacy study design

B

Inhibition of promotes an anti-tumor immune phenotypeC

A SIK3-dependent gene expression pattern is associated with poor surviuval in PRADD

SIK3

/TNF

inde

x

−2

−1

0

●●●● ● ●●● ●● ●●● ●● ●● ●● ●● ●● ●●● ●● ●●●

● ●●● ●● ●● ●●●● ● ● ●●● ●● ● ●● ●● ● ●●● ● ●●● ● ● ●● ●● ●● ●● ●● ●● ●●● ● ●● ●●● ● ●●● ●●● ●● ●● ●●●●● ●●● ●●● ●● ●● ●● ● ●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ●● ●● ● ●●● ●● ●● ●●●● ●● ●●● ●● ● ●●●● ● ●

●

●

●●

●

●

●

●● ●

●

●●

3 5 7 9 11CD8A expression

[RSEM (log2)]

p < 0.01R2 = 0.42

SIK3

/TNF

inde

x

PRF1 / GZMA expression [RSEM (geometric mean, log2)]

−2

−1

0

●

●

●●

●

●

●

●●●

●

●●

●●● ● ● ●●● ●● ● ● ●● ● ●● ● ●● ●●● ● ●●●● ●● ●

● ●●● ●● ●●● ●●● ●● ● ●● ● ●●●● ●● ● ●●●● ●●●● ●●● ●●●● ●●● ● ●●●●● ●●● ●● ●● ●●● ●●● ●● ●● ● ●● ● ● ●●● ● ●● ●● ●● ●● ●●● ● ●● ●● ●● ● ● ●● ●● ●●● ●● ● ● ●●● ●●● ●● ●●●●●● ●● ●●● ●●● ●●●● ● ●

4 6 8

p < 0.01R2 = 0.48

++++++

+++++++++++++

+++++++++++++

++++++ +++++++++

+++++++ + +++++ + ++++ +

+

+

+ +

++ + + + + + + +

0

25

50

75

100

0 1 2 3 4 5 6 7 8

Follow−Up time [Years]

Surv

ival

pro

babi

lity

[%]

p = 0.0012

index low

index high

1 4 8 11 15 180

200

400

600

study days

***Tu

mor

vol

ume

[mm

3 ] vehicle

OMX-0370 qd 100 mg/kg

OMX-0370 bid 100 mg/kg

p=0.06

Group treatment total daily dose[e.g. mg/k/d]

dosing route Cumulated

dosesmice

Analysis

1 vehicle 10 ml/kg QD x 18 p.o 18 10 FC/TG

2 OMX0370 100 mg /kg QD x 18 p.o 18 10 FC/TG

3 OMX0370 100 mg /kg BID x 18 p.o 36 10 FC/TG

Study start: tumor cell injection5x105 MC38 cells (s.c.)

Randomization: TV ~ 100 mm3Treatment start: p.o. 7q7d Last treatment

FACS analysisDay -10

Day 0Day 1 Day 18

vehicle

OMX370 qd

OMX370 bid0

2

4

6

CTL / T reg ratio

ratio

CTL

/ Tre

g

**

*

vehicle

OMX370 qd

OMX370 bid0

10

20

30

40

M2-like TAM(CD206+ MHC-II+)

% o

f CD4

5+ cells

*** ***

vehicle

OMX370 qd

OMX370 bid0

2

4

6

8

cytotoxic T cells(GrzB+)

*

**

% o

f CD4

5+ cells

vehicle

OMX370 qd

OMX370 bid0

2

4

6

cytotoxic T cells(CD25+ CD69+)

**

% o

f CD4

5+ cells

A In vivo e®icacy study design

B

Inhibition of promotes an anti-tumor immune phenotypeC

A SIK3-dependent gene expression pattern is associated with poor surviuval in PRADD

C Inhibition of SIK3 promotes an anti-tumor immune phenotype

D A SIK3-dependent gene expression pattern is associated with poor survival in pancreatic cancer patients

SIK3 inhibition reduces tumor growth in vivo

→→ Novel SIK3 inhibitor OMX-0370 inhibits tumor growth in MC38 murine CRC model

→→ SIK3 inhibitor OMX-0370 induces an anti-tumoral immune phenotype in the tumor microenvironment

→→ Pancreatic cancer patients with a high expression of a TNF/SIK3/NFkB-dependent gene signature have a poor survival prognosis

0.1 1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Murine MC38

HG-9-91-01HG-9-91-01

TNF-+

500 nM1420 nM

1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Human PANC1A B

AS1 inhibition sensitizes a wide array of cell liens to TNF-induced apoptosis

D E

−0.4

-0.2

0

0.2

0.4

HSA

syne

rgy

scor

e

Cancer cell line originbladderbonebrainbreastcolonconnective tissueendometrialhematologicalkidneyliverlungmuscleovarypancreasplacentaprostateskinuterus

HG-9-91-01 + 2 ng/ml TNF on 94 cell lines

sensitive cell lines

resitant cell lines

TNF+HG-91-01

SIK3-inactive

100

50

100

Concentration [nM]

NFkB

act

ivity

[%]

100 1000 10000+-TNF

C

AS1 inhibition reduces HDAC4 phosphorylation andNFkB activity

MC38 PANC1

HG-9-91-01SIK3-inactive

1111 nM 110 nM0.0

0.5

1.0

HDAC

4 ph

osph

oryl

atio

n [%

]no

rmai

leze

d to

DM

SO

*****

A SIK3 inhibition sensitizes a wide array of tumor cell lines to TNF-induced apoptosis

FIGURE 1

A Scheme of the iOTarg platform workflow to identify novel immune checkpoints in pancreatic cancer. Luciferase-positive PANC1 tumor cells were transfected with a siRNA library targeting approx. 3000 genes and co-cultured with patient-derived TILs or medium alone. Tumor viability was measured using luc-based readout. B Gene ranking based on impact on TIL-mediated tumor lysis. Z-scores were calculated using the luciferase activity values from cytotoxicity (with TIlLs) and viability (without TILs) setup. Local regression (LOESS) was used to fit cytotoxicity to viability.C PANC1 tumor cells were transfected with sequence-independent SIK3-specific and control siRNAs and cocultured with T cells. Tumor lysis by T cells was measured and normalized to the respective viability control (addition of medium without TILs).D M579 cells were transfected with SIK3 over expression (OE) or empty vector (EV). T cell-mediated cytotoxicity was assessed as in (B).E siRNA transfected SW480 (left panel) and MCF7 (right panel) were co-cultured with either survivin-specific CTLs or TILs, respectively. M579 (middle panel) were transduced with SIK3-specific shRNA or NTS control and co-cultured as before. Real-time live cell microscopy was used to evaluate tumor cell death using YOYO-1 dye and apoptosis was plotted as area of YOYO-1+ cells/well (µm2/well).

FIGURE 3

A RNA seq analysis showing hierarchical clustering of genes that were differentially regulated in SIK3 knocked down tumor cells after 4 h of TNF treatment. Gene enrichment analysis depictes genes having reduced or missing induction by TNF after SIK3 knockdown (fold change ≥ 2, normalized counts per million > 2, FDR ≤ .05). B Left panel: ATAC sequencing showing chromatin accessibility in single base pair resolution over all differential open chromatin regions that harbor a NFkB or IRF binding motif, respectively. Right panel: Representative IGV genome browser snapshot showing chromatin accessibility data obtained by ATAC-seq at the TRAF1 locus. C ELISA for detection of nuclear p65 subunit of NFκB in siSIK3 vs. siCtrl PANC1 cells treated with TNF.D Rescue of PANC1 cells by double knockdown of SIK3 and HDAC4 before treatment with 100 ng/mL of TNF.

Salt-inducible kinase 3 facilitates tumor cell resistance against cytotoxic T cell attack by shifting TNF signaling from apoptosis to survival

Tillmann Michels1*, Antonio Sorrentino2,3*, Ayse Nur Menevse3, Stefan Bissinger1, Peter Sennhenn1, Valentina Volpin2,3, Sabrina Genssler1, Hannes Loferer1, Christian Kohler4, Rainer Spang3, Michael Rheli3, Christian Schmidl3, Macro Breinig2, Michael Boutros2, Sebastian Meier-Ewert1, Apollon Papadimitriou1, Philipp Beckhove2,3 and Nisit Khandelwal1

1 iOmx Therapeutics, Martinsried/Munich, Germany 2 German Cancer Research Centre (DKFZ), Heidelberg, Germany 3 Regensburg Center for Interventional Immunology (RCI), Regensburg, Germany 4 Institute of Functional Genomics, University of Regensburg, Germany * contributed equally

Research Authors and affiliations

SIK3 mediates intrinsic resistance of tumors against TNF

→→ SIK3-positive tumors were inert to TNF-induced apoptosis→→ Neutralization of TNF or TNFR1 abrogated the apoptosis of SIK3-

negative tumors

siSIK3

siCtrl100

80

60

40

20

0

120

Iso Abanti-TNF

Iso Abanti-TRAIL

Iso Abanti-FasL

Medium

+ activated TIL supernatant -

**

**

** ** ** **

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

Cell

viab

ility

-2 -1 0 1 2 3 40

1.4x107

8.0x106

1.6x107

1.2x107

1.0x107

6.0x106

4.0x106

2.0x106

siCtrl siSIK3

TNF[log(ng/mL)]

0 1 2 3 4 5 6Time (h)

Tum

or C

ell D

eath

(µm

2 /wel

l)

siCtrl

siCtrl

siAS1

siAS1

TNF

+

-

0

5x107

1x108

C

B

A

**

**

Sup. TILsCtrl Ab

anti-TNF

-

--

+

-+

+

+-

+

++-

+

+++-

120

100

80

40

20

0

140

60

siSIK3

D

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

TNFCtrl Ab

anti-TNFR1

-

--

+

--

+

-+

+

+-

0

100

80

40

20

60

*siSIK3

E

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

F

T cell Tumor

PD-1 PDL-1

TCR MHC

TRAIL/FasL

TNF

SIK3

Apoptosis

Intrinsic resistance

Extrinsic resistance

TRAILR/Fas

TNFR1

Schematic representation of extrinsic and intrinsic resistance to TC attack

Blockade of TNF rescues tumor cells from killing

AS1 knockdown mediated tumor lysis is TNF dependentsiSIK3

siCtrl100

80

60

40

20

0

120

Iso Abanti-TNF

Iso Abanti-TRAIL

Iso Abanti-FasL

Medium

+ activated TIL supernatant -

**

**

** ** ** **

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

Cell

viab

ility

-2 -1 0 1 2 3 40

1.4x107

8.0x106

1.6x107

1.2x107

1.0x107

6.0x106

4.0x106

2.0x106

siCtrl siSIK3

TNF[log(ng/mL)]

0 1 2 3 4 5 6Time (h)

Tum

or C

ell D

eath

(µm

2 /wel

l)

siCtrl

siCtrl

siAS1

siAS1

TNF

+

-

0

5x107

1x108

C

B

A

**

**

Sup. TILsCtrl Ab

anti-TNF

-

--

+

-+

+

+-

+

++-

+

+++-

120

100

80

40

20

0

140

60

siSIK3

D

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

TNFCtrl Ab

anti-TNFR1

-

--

+

--

+

-+

+

+-

0

100

80

40

20

60

*siSIK3

E

Cell

viab

ility

[%]

(nor

mal

ized

to co

ntro

l)

F

T cell Tumor

PD-1 PDL-1

TCR MHC

TRAIL/FasL

TNF

SIK3

Apoptosis

Intrinsic resistance

Extrinsic resistance

TRAILR/Fas

TNFR1

Schematic representation of extrinsic and intrinsic resistance to TC attack

Blockade of TNF rescues tumor cells from killing

AS1 knockdown mediated tumor lysis is TNF dependent

B Blockade of TNF rescues SIK3-deficient tumor cells from apoptosis

SIK3-induced immune resistance is TNF-dependent

C D E

SIK3 promotes NFkB transcription of pro-survival genes by inhibiting HDAC4

→→ SIK3 enhanced NFkB-mediated gene expression in tumor cells upon TNF treatment

→→ Furthermore, SIK3 regulates NFkB activity at an epigenomic level→→ Abrogation of HDAC4 is involved in SIK3-mediated immune

suppression

FIGURE 2

A Scheme representing tumor extrinsic (direct T cell inhibition) versus intrinsic (inherent resistance to soluble effector molecules) immune resistance pathways. B PANC1-luc cells were transfected with indicated siRNAs and subjected to the supernatant of polyclonally-activated TILs. Neutralization of the effector molecules in the supernatant was done using anti-TNF, anti-TRAIL or anti-FasL (Ab) antibodies or matched isotype controls (Iso). C Dose-response effect of TNF treatment on the viability of SIK3 knocked down PANC1-luc cells compared to control cells in the luc-based cytotoxicity assay. D Supernatant from polyclonally stimulated T cells was incubated with 100 (+), 300 (++) or 900 (+++) ng/mL of anti-TNF neutralizing antibody before addition to SIK3 knocked down (siSIK3) PANC1 luc cells. Isotype control (Ctrl Ab) was used at concentration of 900 ng/mL. Data was normalized to siCtrl. E Effect of TNFR1 blockade on SIK3 knocked down PANC1 cells after TNF treatment.

−2 0 2Z score

+ +– –

siSIK3siCtrl+ +– – TNF

EnrichrTRANSFAC/JASPAR PWMs adj. P

GO Term: Biological Process adj. P

RELA (human) 4.9 x 10-6

NFKB1 (human) 0,00013

regulation of cell activation 5.0 x 10-7

reg. of immune e�ector process 1.2 x 10-6

reg. of leukocyte proliferation 1.3 x 10-6

reg. of cytokine production 1.4 x 10-6

reg. of lymphocyte di�erentiation 1.4 x 10-6

205 Genes

siSIK3

siSIK3siCtrl

siCtrl

30 min15 minUnstimulated

+ TNF

**

Activ

e nu

clea

r NFκ

BFo

ld ch

ange

4

3

2

1

0 siCtrl siSIK3

* *179%

Tum

or ce

ll su

rviv

al

0

1x106

2x106

3x106

4x106

TNFsiCtrl

siHDAC4siSIK3

++--

+-+-

+--+

+-++

A Knockdown of AS1 reduces NFkB-dependent gene expression upon TNF treatment

B Knockdown of AS1 alters chromatin accessibility in NFkB-dependent genes

C TNF induced NFkB activity depends on AS1 D Knockdown of HDAC4 rescues AS1-deficiant cells

0

5

10

15

20

25

0

5

10

15

20

25

−1000 −500 0 500 1000

NFkB

0

5

10

15

0

5

10

15

−1000 −500 0 500 1000

IRF

Distance from motif center (base pairs)

Norm

aliz

ed A

TAC-

seq

read

coun

ts TNF4 h

24 h

siCtrl (1)siCtrl (2)

siSIK3 (1)siSIK3 (2)

TRAF1

Genomic region

A Knockdown of SIK3 reduces NFkB-dependent gene expression upon TNF treatment

−2 0 2Z score

+ +– –

siSIK3siCtrl+ +– – TNF

EnrichrTRANSFAC/JASPAR PWMs adj. P

GO Term: Biological Process adj. P

RELA (human) 4.9 x 10-6

NFKB1 (human) 0,00013

regulation of cell activation 5.0 x 10-7

reg. of immune e�ector process 1.2 x 10-6

reg. of leukocyte proliferation 1.3 x 10-6

reg. of cytokine production 1.4 x 10-6

reg. of lymphocyte di�erentiation 1.4 x 10-6

205 Genes

siSIK3

siSIK3siCtrl

siCtrl

30 min15 minUnstimulated

+ TNF

**

Activ

e nu

clea

r NFκ

BFo

ld ch

ange

4

3

2

1

0 siCtrl siSIK3

* *179%

Tum

or ce

ll su

rviv

al

0

1x106

2x106

3x106

4x106

TNFsiCtrl

siHDAC4siSIK3

++--

+-+-

+--+

+-++

A Knockdown of AS1 reduces NFkB-dependent gene expression upon TNF treatment

B Knockdown of AS1 alters chromatin accessibility in NFkB-dependent genes

C TNF induced NFkB activity depends on AS1 D Knockdown of HDAC4 rescues AS1-deficiant cells

0

5

10

15

20

25

0

5

10

15

20

25

−1000 −500 0 500 1000

NFkB

0

5

10

15

0

5

10

15

−1000 −500 0 500 1000

IRF

Distance from motif center (base pairs)

Norm

aliz

ed A

TAC-

seq

read

coun

ts TNF4 h

24 h

siCtrl (1)siCtrl (2)

siSIK3 (1)siSIK3 (2)

TRAF1

Genomic region

B Knockdown of SIK3 decreases chromatin accesibility in NFkB-dependent genes

FIGURE 5

A Treatment schedule of an in vivo efficacy study for SIK3 inhibitor OMX-0370. B MC38 cells (5x105; s.c.) were implanted in C57BL/6 mice and treated according to A. Statistical significance was calculated using a two-way ANOVA analysis including Tukeys multiple comparison analysis. C Intra tumoral immune infiltrate was analyzed after the last treatment using flow cytometry. All cell populations were calculated as the percentage of intratumoral CD45+ cells. Statistical significance was calculated with one-way ANOVA analysis including Tukeys multiple comparison analysis.D Using differential gene expression shown in Fig 3A, a gene signature (SIK3/TNF index) was generated on the pancreatic adenocarcinoma dataset from TCGA (n=185). This index was correlated to either CD8A expression (left panel) or the geometric mean of PRF1 and GZMA (marker for T cell activity; middle panel). Patients were separated into index high or low (based on a quantile-quantile plot; indicated by colors). Patient survival was separated using the SIK3/TNF index (right panel).

PANC1 : TIL#1

siCtrl s1 s2 s3

siPD-L1

0

25

50

75

100

rem

aini

ng tu

mor

cell

viab

ility

[%]

********

****

****

siSIk3

M579 : TIL209

MelanomaM579 : TIL209

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Colorectal cancerSW480 : TIL#1

Tum

or ce

ll dea

th (µ

m2 /w

ell)

Time (h)

Breast cancerMCF7 : survivin-TCs

siCtrl

siCtrl

siSIK3

siSIk3

TCs

+

-

Tum

or ce

ll dea

th (µ

m2 /w

ell)

0 15 300

5x105

1x106

0 20 400

5x106

1x107

Time (h)0 10 20 30 40 50 60 70

0

1x106

2x106

3x106

4x106

5x106

shCtrl

shCtrl

shSIK3

shSIK3

TCs

+

-

siCtrl

siCtrl

siSIK3

siSIK3

TCs

+

-

0

25

50

75

100

*

EV OE

rem

aini

ng tu

mor

cell

viab

ility

[%]

A iOtarg screening overview

siRNA validation

AS1 Target validation in solid cancers

E Overexpression of AS1 rescues tumor cells from TC-lysis

Pancreatic cancer screening results

−4

−2

0

2

4

6

0 500 1000 1500 2000 2500 3000Gene rank

TC-m

edia

ted

lysi

s (LO

ESS

scor

e)

siSIK3

siCEACAM6siPD-L1

siCtrl

Potential immunemodulators

D

B C

Ag-specific MIL cell culture

Tumor cell culture

Luciferase

Transfection

Co-culture

Lysis

Residual luciferase

Luci

fera

se U

nits

Luci

fera

se U

nits

Control si

RNA

Immune-checkpoint

siRNA

Control si

RNA

Immune-checkpoint

siRNA

Read-out

Cytotoxicity setup Viability setup

Screening

D SIK3 overexpression rescues tumor cells from TIL-lysis

SIK3 inhibition reduces NFkB activity and HDAC4 phosphorylation

B

C

FIGURE 4

AA panel of 94 human tumor cell lines were treated with 2 ng/ml TNF and titration (1 µM- 1 nM) of HG-9-91-01 or DMSO control for 120 h. Growth was measured as optical density using the SRB method. Dose-response curves were fitted to the data (non-linear regression) and the highest single agent (HSA) model was used to calculate synergy effects. Synergy and resistance to the combination of TNF and HG-9-91-01 are shown in the waterfall plot.B NFkB-luc reporter MC38 clones were treated with different concentrations of HG-9-91-01 or SIK3-inactive control compound before addition of 10 ng/ml murine TNF. NF-kB activity was determined using luminescence.C Phosphorylated HDAC4 levels of PANC1 cells treated with different concentrations of HG-9-91-01 orSIK3-inactive control compound were analyzed in Meso Scale Discovery (MSD)-based assay with anti-HDAC4 capture and anti-pHDAC4 detection antibodies. Data is shown as percent (%) of phosphorylation remaining normalized to DMSO control.

0.1 1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Murine MC38

HG-9-91-01HG-9-91-01

TNF-+

500 nM1420 nM

1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Human PANC1A B

AS1 inhibition sensitizes a wide array of cell liens to TNF-induced apoptosis

D E

−0.4

-0.2

0

0.2

0.4

HSA

syne

rgy

scor

e

Cancer cell line originbladderbonebrainbreastcolonconnective tissueendometrialhematologicalkidneyliverlungmuscleovarypancreasplacentaprostateskinuterus

HG-9-91-01 + 2 ng/ml TNF on 94 cell lines

sensitive cell lines

resitant cell lines

TNF+HG-91-01

SIK3-inactive

100

50

100

Concentration [nM]

NFkB

act

ivity

[%]

100 1000 10000+-TNF

C

AS1 inhibition reduces HDAC4 phosphorylation andNFkB activity

MC38 PANC1

HG-9-91-01SIK3-inactive

1111 nM 110 nM0.0

0.5

1.0

HDAC

4 ph

osph

oryl

atio

n [%

]no

rmai

leze

d to

DM

SO

*****

0.1 1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Murine MC38

HG-9-91-01HG-9-91-01

TNF-+

500 nM1420 nM

1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Human PANC1A B

AS1 inhibition sensitizes a wide array of cell liens to TNF-induced apoptosis

D E

−0.4

-0.2

0

0.2

0.4

HSA

syne

rgy

scor

e

Cancer cell line originbladderbonebrainbreastcolonconnective tissueendometrialhematologicalkidneyliverlungmuscleovarypancreasplacentaprostateskinuterus

HG-9-91-01 + 2 ng/ml TNF on 94 cell lines

sensitive cell lines

resitant cell lines

TNF+HG-91-01

SIK3-inactive

100

50

100

Concentration [nM]

NFkB

act

ivity

[%]

100 1000 10000+-TNF

C

AS1 inhibition reduces HDAC4 phosphorylation andNFkB activity

MC38 PANC1

HG-9-91-01SIK3-inactive

1111 nM 110 nM0.0

0.5

1.0

HDAC

4 ph

osph

oryl

atio

n [%

]no

rmai

leze

d to

DM

SO

*****

0.1 1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Murine MC38

HG-9-91-01HG-9-91-01

TNF-+

500 nM1420 nM

1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Human PANC1A B

AS1 inhibition sensitizes a wide array of cell liens to TNF-induced apoptosis

D E

−0.4

-0.2

0

0.2

0.4

HSA

syne

rgy

scor

e

Cancer cell line originbladderbonebrainbreastcolonconnective tissueendometrialhematologicalkidneyliverlungmuscleovarypancreasplacentaprostateskinuterus

HG-9-91-01 + 2 ng/ml TNF on 94 cell lines

sensitive cell lines

resitant cell lines

TNF+HG-91-01

SIK3-inactive

100

50

100

Concentration [nM]

NFkB

act

ivity

[%]

100 1000 10000+-TNF

C

AS1 inhibition reduces HDAC4 phosphorylation andNFkB activity

MC38 PANC1

HG-9-91-01SIK3-inactive

1111 nM 110 nM0.0

0.5

1.0

HDAC

4 ph

osph

oryl

atio

n [%

]no

rmai

leze

d to

DM

SO

*****

0.1 1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Murine MC38

HG-9-91-01HG-9-91-01

TNF-+

500 nM1420 nM

1 10 100 1000 100000

50

100

Concentration [nM]

Viab

ility

[%]

Human PANC1A B

AS1 inhibition sensitizes a wide array of cell liens to TNF-induced apoptosis

D E

−0.4

-0.2

0

0.2

0.4

HSA

syne

rgy

scor

e

Cancer cell line originbladderbonebrainbreastcolonconnective tissueendometrialhematologicalkidneyliverlungmuscleovarypancreasplacentaprostateskinuterus

HG-9-91-01 + 2 ng/ml TNF on 94 cell lines

sensitive cell lines

resitant cell lines

TNF+HG-91-01

SIK3-inactive

100

50

100

Concentration [nM]

NFkB

act

ivity

[%]

100 1000 10000+-TNF

C

AS1 inhibition reduces HDAC4 phosphorylation andNFkB activity

MC38 PANC1

HG-9-91-01SIK3-inactive

1111 nM 110 nM0.0

0.5

1.0

HDAC

4 ph

osph

oryl

atio

n [%

]no

rmai

leze

d to

DM

SO

*****

−2 0 2Z score

+ +– –

siSIK3siCtrl+ +– – TNF

EnrichrTRANSFAC/JASPAR PWMs adj. P

GO Term: Biological Process adj. P

RELA (human) 4.9 x 10-6

NFKB1 (human) 0,00013

regulation of cell activation 5.0 x 10-7

reg. of immune e�ector process 1.2 x 10-6

reg. of leukocyte proliferation 1.3 x 10-6

reg. of cytokine production 1.4 x 10-6

reg. of lymphocyte di�erentiation 1.4 x 10-6

205 Genes

siSIK3

siSIK3siCtrl

siCtrl

30 min15 minUnstimulated

+ TNF

**

Activ

e nu

clea

r NFκ

BFo

ld ch

ange

4

3

2

1

0 siCtrl siSIK3

* *179%

Tum

or ce

ll su

rviv

al

0

1x106

2x106

3x106

4x106

TNFsiCtrl

siHDAC4siSIK3

++--

+-+-

+--+

+-++

A Knockdown of AS1 reduces NFkB-dependent gene expression upon TNF treatment

B Knockdown of AS1 alters chromatin accessibility in NFkB-dependent genes

C TNF induced NFkB activity depends on AS1 D Knockdown of HDAC4 rescues AS1-deficiant cells

0

5

10

15

20

25

0

5

10

15

20

25

−1000 −500 0 500 1000

NFkB

0

5

10

15

0

5

10

15

−1000 −500 0 500 1000

IRF

Distance from motif center (base pairs)

Norm

aliz

ed A

TAC-

seq

read

coun

ts TNF4 h

24 h

siCtrl (1)siCtrl (2)

siSIK3 (1)siSIK3 (2)

TRAF1

Genomic region

−2 0 2Z score

+ +– –

siSIK3siCtrl+ +– – TNF

EnrichrTRANSFAC/JASPAR PWMs adj. P

GO Term: Biological Process adj. P

RELA (human) 4.9 x 10-6

NFKB1 (human) 0,00013

regulation of cell activation 5.0 x 10-7

reg. of immune e�ector process 1.2 x 10-6

reg. of leukocyte proliferation 1.3 x 10-6

reg. of cytokine production 1.4 x 10-6

reg. of lymphocyte di�erentiation 1.4 x 10-6

205 Genes

siSIK3

siSIK3siCtrl

siCtrl

30 min15 minUnstimulated

+ TNF

**

Activ

e nu

clea

r NFκ

BFo

ld ch

ange

4

3

2

1

0 siCtrl siSIK3

* *179%

Tum

or ce

ll su

rviv

al

0

1x106

2x106

3x106

4x106

TNFsiCtrl

siHDAC4siSIK3

++--

+-+-

+--+

+-++

A Knockdown of AS1 reduces NFkB-dependent gene expression upon TNF treatment

B Knockdown of AS1 alters chromatin accessibility in NFkB-dependent genes

C TNF induced NFkB activity depends on AS1 D Knockdown of HDAC4 rescues AS1-deficiant cells

0

5

10

15

20

25

0

5

10

15

20

25

−1000 −500 0 500 1000

NFkB

0

5

10

15

0

5

10

15

−1000 −500 0 500 1000

IRF

Distance from motif center (base pairs)

Norm

aliz

ed A

TAC-

seq

read

coun

ts TNF4 h

24 h

siCtrl (1)siCtrl (2)

siSIK3 (1)siSIK3 (2)

TRAF1

Genomic region

D Knockdown of HDAC4 rescues SIK3-deficiant cells

C TNF-induced NFkB activity depends on SIK3