Mutant Cancers 2 3 Heinz Hammerlindl1*, Dinoop Ravindran Menon1*, Sabrina Hammerlindl1, Abdullah Al

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Author Manuscript Published OnlineFirst on December 1, 2017; DOI: 10.1158/1078-0432.CCR-16-2118 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Hammerlindl et. al

1 Acetylsalicylic Acid Governs the Effect of Sorafenib in RAS Mutant Cancers 2 3 Heinz Hammerlindl1*, Dinoop Ravindran Menon1*, Sabrina Hammerlindl1, Abdullah Al

4 Emran1, Joachim Torrano1, Katrin Sproesser3, Divya Thakkar1, Min Xiao3, Victoria G.

5 Atkinson5, Brian Gabrielli4, Nikolas K. Haass2, Meenhard Herlyn3, Clemens Krepler3, Helmut

6 Schaider1,2†

8 1Dermatology Research Centre, The University of Queensland, The University of

9 Queensland Diamantina Institute, Translational Research Institute, Brisbane, Australia;

10 2The University of Queensland, The University of Queensland Diamantina Institute,

11 Translational Research Institute, Brisbane, Australia;

12 3The Wistar Institute, Philadelphia, PA, U.S.A.;

13 4Mater Medical Research Institute, The University of Queensland, Translational Research

14 Institute, Brisbane, Australia;

15 5Division of Cancer Services, Princess Alexandra Hospital, Brisbane, Australia;

16 *These authors contributed equally to the study 17

18 Running title:

19 Combined aspirin and sorafenib for RAS-mutant cancer therapy

21 Key words:

22 Melanoma, Lung Cancer, NRAS, Sorafenib, Aspirin, RAS, ERK, AMPK

Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2017 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 1, 2017; DOI: 10.1158/1078-0432.CCR-16-2118 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Hammerlindl et. al

2 Grant Support

3 This work was funded by the Epiderm Foundation (H.S.), the Princess Alexandra Hospital

4 Research Foundation (PARSS2016_NearMiss) (H.S.), NIH grants PO1 CA114046, P50

5 CA174523, and the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (both

6 M.H.). A.A.E. is funded by The University of Queensland International Scholarship (UQI);

7 H.H. is funded by the International Postgraduate Research Scholarship (IPRS) and UQ

8 Centennial Scholarship (UQCent). N.K.H. is funded by the National Health and Medical

9 Research Council (APP1084893).

11 †Corresponding author

12 Helmut Schaider, MD, FACD

13 The University of Queensland Diamantina Institute

14 Translational Research Institute

15 The University of Queensland

16 37 Kent Street

17 Woolloongabba QLD 4102

18 Australia

19 T 61 7 3443 7395

20 F 61 7 3443 7799

21 E-mail: [email protected]

24 Conflict of interest

25 The authors declare no conflict of interest

Hammerlindl et. al

1 Abstract

2 Purpose: Identify and characterize novel combinations of sorafenib with anti-inflammatory

3 painkillers to target difficult to treat RAS-mutant cancer.

4 Experimental Design: The cytotoxicity of acetylsalicylic acid (aspirin) in combination with

5 the multikinase inhibitor sorafenib (Nexavar®) was assessed in RAS-mutant cell lines in vitro.

6 The underlying mechanism for the increased cytotoxicity was investigated using selective

7 inhibitors and shRNA-mediated gene knockdown. In vitro results were confirmed in RAS-

8 mutant xenograft mouse models in vivo.

9 Results: The addition of aspirin but not isobutylphenylpropanoic acid (ibruprofen®) or

10 celecoxib (celebrex®) significantly increased the in vitro cytotoxicity of sorafenib.

11 Mechanistically, combined exposure resulted in increased BRAF/CRAF dimerization and the

12 simultaneous hyper-activation of the AMPK and ERK pathways. Combining sorafenib with

13 other AMPK activators, like metformin or A769662, was not sufﬁcient to decrease cell

14 viability due to sole activation of the AMPK pathway. The cytotoxicity of sorafenib and aspirin

15 was blocked by inhibition of the AMPK or ERK pathways through shRNA or via

16 pharmacological inhibitors of RAF (LY3009120), MEK (trametinib) or AMPK (compound C).

17 The combination was found to be specific for RAS/RAF-mutant cells and had no signiﬁcant

18 effect in RAS/RAF-wild type keratinocytes or melanoma cells. In vivo treatment of human

19 xenografts in NSG mice with sorafenib and aspirin significantly reduced tumor volume

20 compared to each single-agent treatment alone.

21 Conclusion: Combined sorafenib and aspirin exerts cytotoxicity against RAS/RAF-mutant

22 cells by simultaneously affecting two independent pathways and represents a promising

23 novel strategy for the treatment of RAS-mutant cancers.

28 3

Hammerlindl et. al

1 Translational Relevance

2 To date, no therapies directly targeting mutant RAS have been approved, leaving

3 chemotherapy with very low response rates or immunotherapy as the only treatment options

4 for RAS-mutant cancers. Here we report a novel strategy to target RAS-mutant cancer,

5 especially NRAS-mutant melanoma, by combining the multi-kinase inhibitor sorafenib and

6 the non-steroidal anti-inflammatory drug acetylsalicylic acid (aspirin), both of which are

7 clinically approved and tested. The addition of aspirin strongly enhanced the in vitro and in

8 vivo cytotoxicity of otherwise ineffective sorafenib dosages. The combination of sorafenib

9 and aspirin but no other AMPK activators simultaneously induced activation of the AMPK and

10 ERK pathways, which are both necessary for drug effectivity. This finding suggests that

11 combining sorafenib with aspirin could be a viable treatment strategy for RAS-mutant

12 cancers including NRAS-mutant melanoma.

Hammerlindl et. al

1 Introduction

2 Mutant neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) was the first oncogene

3 identified in melanoma (1) and it is now known that approximately 20% of all melanomas

4 harbor mutations in NRAS, 2% in KRAS and 1% in HRAS (2). While KRAS and HRAS only

5 play a minor role for melanoma, KRAS in particular is frequently mutated in other cancers,

6 including lung, colon and pancreatic carcinomas (3). Mechanistically, RAS proteins are

7 GTPases that activate downstream signaling pathways involved in proliferation and cell

8 survival upon GTP binding (3). Genetic mutations are located in codons 12, 13 and 61, and

9 more than 80% of mutant NRAS harbor a mutation at codon 61 (4). Mutations result in

10 reduced GTPase activity, which causes preferential binding of GTP and therefore constitutive

11 activation of RAS signaling (5). In recent years the advent of targeted therapies has

12 advanced melanoma treatment but focused on mutant BRAF, whereas no strategies directly

13 targeting mutant NRAS have been approved (6,7). Inhibitors for RAS are particularly difficult

14 to develop (8,9) leaving low response chemotherapy (10) or immunotherapy (11) with high

15 toxicity rates as therapeutic strategies for patients with NRAS-mutant melanoma. Attempts to

16 directly target RAS mutations include farnesyl transferase inhibitors, which are supposed to

17 prevent posttranslational modifications required for the integration of RAS into the plasma

18 membrane (12) thus preventing the interaction of RAS and the prenyl-binding protein PDEδ

19 (13). Unfortunately, farnesyl transferase inhibitors showed disappointing results in clinical

20 settings (12) and inhibitors of PDEδ binding to farnesylated KRAS still require more

21 development to optimize the drugs (14). Other attempts to target RAS-mutant cancers

22 include blocking RAS downstream targets. Inhibition of MEK (6,15) or MEK in combination

23 with PI3K/mTOR inhibitors have shown promising results (10). However, inhibition of MEK

24 leads to the development of resistance, similar to strategies in BRAF-mutant melanoma (16).

25 Several mechanisms of resistance have been proposed for NRAS-mutant melanoma

26 including PDGF receptor β signaling (17) emphasizing the importance of novel single or

27 combination therapies for sustained treatment. Sorafenib (Nexavar, BAY 43–9006; Bayer

28 Healthcare Pharmaceuticals) is a multikinase inhibitor that targets both C-RAF and B-RAF as 5

Hammerlindl et. al

1 well as the vascular endothelial growth factor receptor family (VEGFR-2 and VEGFR-3) and

2 platelet-derived growth factor receptor family (PDGFR-) (18), among others. Sorafenib is

3 FDA-approved for the treatment of advanced renal cell carcinoma and patients with

4 unresectable hepatocellular cancer (19) with most common adverse events being skin

5 rashes, diarrhea, and alopecia (20). Additionally the development of squamous cell

6 carcinomas and keratoacanthomas has been reported (21,22). These side effects resulted in

7 patients requiring dose reductions, interrupting or discontinuing therapy raising concerns

8 about the toxicity, efficacy and safety of sorafenib (20). One strategy to overcome drug-

9 induced toxicity is to combine sorafenib with other drugs to reduce the effective dose

10 required to trigger a tumor specific response without inducing systemic toxicity. Several

11 studies have explored possible anticancer effects of sorafenib in combination with other

12 targeted inhibitors or radiation, which showed limited efficacy (reviewed in (23)).

13 Non-steroidal anti-inflammatory drugs (NSAIDs) have been reported to reduce overall cancer

14 risk including prostate (24), colorectal (25) and skin cancer (26). The association between

15 NSAIDs and melanoma risk is less clear, and several studies yielded conflicting results (27).

16 Acetylsalicylic acid (Aspirin) is an intriguing agent as it is one of the most widely used drugs.

17 Synergistic effects of sorafenib and aspirin have already been described in RAS wild type

18 hepatocellular carcinoma where it has been reported to reduce the pro-metastatic effect of

19 sorafenib monotherapy (28).

20 Here we show that combined sorafenib and aspirin resulted in synergistic cytotoxicity in

21 RAS/RAF-mutant cancers including NRAS-mutant melanoma by simultaneously activating

22 the AMPK and ERK pathways. The combined treatment, which is effective in vivo, allows the

23 concentration of sorafenib to be substantially reduced with the likelihood of less tissue

24 toxicity. These data provide a rationale for the application of this combination in RAS-mutant

25 cancer patients.

Hammerlindl et. al

2 Materials and methods

4 Cell culture

5 KRAS-mutant human lung adenocarcinoma cell lines A549 and H358 as well as RAS/RAF-

6 wild type immortalized human keratinocytes HaCaT were kindly provided Dr. Gerald Hoefler

7 (Institute of Pathology, Medical University of Graz, Graz, Austria). The RAS/RAF-wild type

8 human breast cancer cell line SkBr3 was kindly provided by Dr Fiona Simpson (The

9 University of Queensland Diamantina Institute, Brisbane, QLD). All cell lines are routinely

10 tested for mycoplasma as described previously (29,30) and were authenticated in 2016 by

11 the analytical facility of the QIMR Berghofer Medical Research Institute (Brisbane, Australia)

12 via STR fingerprinting. All experiments were performed within 3 month after thawing the

13 respective cell lines. Cells were grown in RPMI-1640 medium (Sigma-Aldrich), supplemented

14 with 5% fetal bovine serum (Assay Martrix) and 2% L-glutamine (Life technologies) and

15 maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells were harvested for

16 individual experiments after washing with PBS (pH 7,4) (Life Technologies) using trypsin

17 (Life Technologies).

19 Viral vector transduction

20 Lentiviral vectors containing shRNA targeting AMPK1/2 (shPRKAA1, NM_006251, CloneID

21 TRCN0000000861; shPRKAA2 NM_006252, CloneID TRCN0000002171), or BRAF

22 (NM_004333, CloneID TRCN0000231130) were purchased from Sigma-Aldrich. Cells were

23 prepared in 12-well plates to reach confluence of 50-80%, pretreated with 8 µg/ml polybrene

24 (Sigma-Aldrich) for 2 h followed by the addition of 25 µL of the viral supernatant. Transduced

25 cells were subjected to selection with puromycin (Sigma-Aldrich) at a concentration of 5

26 µg/ml 72 h post transfection. Cells were then maintained in medium containing 1.5 µg/ml.

Hammerlindl et. al

2 Immunoblotting

3 Whole-cell lysates were generated using RIPA buffer (Sigma-Aldrich) supplemented with 1%

4 protease inhibitor cocktail (Active Motive). Protein lysates from frozen tissue samples were

5 generated using 500µL RIPA buffer per 10mg of tissue and sonicated at 180 watts 10x10

6 seconds. The protein concentration was measured using Bradford Protein Assay (BioRad).

7 15 µg of protein were separated on a 6-10% SDS-polyacrylamide gel followed by transfer to

8 a polyvinylidene difluoride membrane (BioRad). The membrane was then probed for the

9 protein of interest with the specific primary and the corresponding peroxidase conjugated

10 secondary antibodies (Supplementary Table 1). Proteins were visualized using Amersham

11 ECL Prime Western Blotting Detection Reagent (GE Healthcare) and scanned on an LI-COR

12 C-DiGit® Blot Scanner. The membranes were stripped and re-probed using Restore Plus

13 Western Blot stripping buffer (Life technologies) as required in the individual experiments.

14 Immunoblots were quantified using ImageJ and the ratio of phosphorylated to total protein

15 was calculated and normalized to control of the same immunoblot.

17 Immunoprecipitation.

18 Cells were exposed to drugs for 24 hours as indicated in the respective experiments, lysed

19 using 1X Cell Lysis Buffer (Cell Signaling) and sonicated on ice three times for 5 seconds.

20 Cell lysate was subject to a pre-cleaning step by incubating with Protein G Agarose Beads

21 (Cell Signaling) for 1 hour at 4ºC with gentle rocking followed by the addition of 2μL of c-Raf-

22 1 antibody (BD Transduction Laboratories™) and incubated overnight at 4ºC.

23 Immunoprecipitation was performed by adding 10µL Protein G Agarose Beads (Cell

24 Signaling) per 100μL cell lysate and incubated for 3 hours. Co-immunoprecipitation was then

25 assessed by immunoblotting using specific antibodies for CRAF and BRAF (Supplementary

26 Table 1)

Hammerlindl et. al

3 Inhibitors

4 Sorafenib, trametinib, dabrafenib and LY3009120 were purchased from Selleck Chemicals.

5 Dorsomorphin (compound C), A-769662, SC-560 and metformin were purchased from

6 Cayman Chemical. Celecoxib and isobutylphenylpropanoic acid were purchased from Sigma

7 Aldrich.

9 Flow cytometry

10 Caspase 3 activation was assessed using the Active Caspase 3 apoptosis kit (BD

11 Biosciences) following the manufacturers protocol. The samples were analysed with a BD

12 ACCURI C6 PLUS from the Translational Research Institute (TRI) FACS core facility.

14 MTT assay

15 1x104 cells were seeded in 96-well culture plates for allocated times as mentioned in the

16 experiments. Cells were treated with drugs depending on the experimental setup 48 hours

17 after the initial seeding and subsequently incubated with MTT (3-(4, 5-dimethylthiazolyl-2)-

18 2,5 diphenyltetrazolium bromide, Thermo Fisher Scientific) reagent (1/10 dilution in full

19 growth medium) at 37ºC for 4 h. Following incubation, 100 µl DMSO was added and

20 incubated for 10 minutes in the incubator. Absorbance was measured at 540 nm using a

21 microtiter plate reader. All experiments were done in triplicate or duplicate and a final

22 concentration of 1% DMSO was used as control.

24 Cell survival crystal violet staining

25 Cells, which have been exposed to drugs for various time points, were fixed with 4%

26 paraformaldehyde, followed by 30 min incubation with 0.1% crystal violet in 4%

27 paraformaldehyde. The plates were washed and imaged using a Chemi Doc TM XRS

28 Universal Hood (Bio-Rad). A final concentration of 1% DMSO was used as control.

Hammerlindl et. al

3 Drug synergy assessment

4 Cell viability was assessed using the alamarBlue assay and synergy was assessed using the

5 Bliss independence model as previously described (31). Briefly, data was normalized to

6 doxorubicin and DMSO controls and converted to fraction-affected values (F). Next, the

7 predicted inhibition values (P) were calculated: [P=Fa + Fb – Fab 0

1]. Predicted F equals

8 the fraction affected by compound “a” (Fa) at concentration x plus the fraction affected by

9 compound “b” (Fb) at concentration y minus the product of the two (Fab). The difference

10 between the predicted additive fraction affected and the experimentally observed fraction

11 affected is the Bliss number. Positive value indicates synergy, a negative value indicates

12 antagonism and an overlap of predicted and observed combination effects gives a Bliss

13 number of zero and indicates additivity. Due to the nature of the assay, we detect a

14 background noise level of +/- 15%.

16 In vivo study

17 All animal experiments were performed in accordance with institutional guidelines under

18 Wistar IACUC protocol 111954 in NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Lab)

19 mice or in accordance with institutional guidelines of UQ animal ethics committee ethics

20 number: SOM/TRI/197/15/DRC in C.B-17/IcrHanHsdArcPrkdcscid mice (Animal Resources

21 Centre Canning Vale, Australia). Animals were inoculated s.c. with 1x106 human WM1366

22 melanoma cells in a 100 ul suspension of matrigel (BD Matrigel™) / complete media at a

23 ratio of 1:1 or with 2.5x106 human A549 lung adenocarcinoma cells suspended in complete

24 media. After tumors had reached a volume of 100-200 mm3, mice were randomized into

25 groups of 4 mice as indicated in the respective experiments: vehicle (0.75% hydroxyl methyl

26 cellulose/25% ethanol / 10% DMSO), sorafenib 30mg/kg or 15mg/kg, aspirin 100mg/kg or

27 200mg/kg, metformin 100mg/kg, sorafenib + aspirin and sorafenib + metformin. Tumor size

28 was assessed multiple times per week using a caliper. Animals were sacrificed at the

Hammerlindl et. al

1 experiment end point or when tumor volume exceeded the ethical limit. Tumors were then

2 harvested, snap frozen in liquid nitrogen and stored at -80ºC until further processing.

3 Immunohistochemistry

4 Tumors were formalin-fixed, paraffin-embedded and stained using a Discovery Ultra Ventana

5 in the TRI Histology Core Facility. Deparaffinsation and antigen retrieval was performed

6 before using pre-diluted antibodies (Supplementary Table 1) or rabbit IgG as negative control

7 (Invitrogen). H&E staining was performed according to common methods. Staining intensity

8 was assessed and immunoreactivity was calculated as previously described (32). Slides

9 were scanned using the Olympus VS120 slide scanner (20x).

11 Statistical analysis

12 Represented data are expressed as arithmetic mean +/- standard deviation of three

13 independent experiments. Unpaired t-test has been used to determine statistical significance.

14 N.s. indicates a P value > 0.05, * indicates a P value ≤ 0.05, ** indicates a P value ≤ 0.01, ***

15 indicates a P value ≤ 0.001

17 Results

18 Sorafenib and aspirin synergistically induce cell death in NRAS-mutant melanoma

19 The NRASQ61K-mutant melanoma cell line WM1366 was non-responsive to sorafenib at low

20 concentrations (0.25-1 µM) but showed an almost complete loss of viability at 5 µM (Figure

21 1A). Exposure to aspirin alone at concentrations up to 2 mM did not affect cell viability

22 (Figure 1A). The combination of sorafenib and aspirin, however, showed a dose-dependent

23 toxicity, with effects of the combination observed with sorafenib concentrations as low as

24 250nM and 2mM aspirin (Figure 1B). The efficacy of this combination was confirmed by

25 MTT assay, which showed a significant reduction of cell proliferation after 72 h of drug

26 exposure compared to single exposure (Supplementary Figure 1A). Next, we investigated

27 the combination of sorafenib and aspirin using the Bliss Independence Model (33) and found

Hammerlindl et. al

1 synergistic efficacy in a wide range of combined concentrations of both drugs (Figure 1C).

2 The combination of sorafenib and aspirin induced apoptosis at a similar level to sorafenib

3 5µM, indicated by the level of active caspase 3 (Figure 1D). The efficacy of combined

4 sorafenib and aspirin was confirmed in three other NRAS-mutant melanoma cell lines

5 (Supplementary Figure 1B). Combining sorafenib with other NSAIDs, the specific COX2

6 (PTGS2) inhibitor celecoxib, the specific COX1 (PTGS1) inhibitor SC-560 or the non-specific

7 COX1 and COX2 inhibitor isobutylphenylpropanoic acid (Ibuprofen) showed no synergistic

8 toxicity (Supplementary Figure 1C, D and 1E), suggesting that the effect of sorafenib and

9 aspirin is independent of the known aspirin targets COX1 and COX2. Taken together,

10 sorafenib at low concentrations in combination with aspirin synergistically induces apoptosis

11 in NRAS-mutant melanoma independently of COX1 and COX2 inhibition.

13 KRAS- and BRAF-mutant but not RAS/RAF-wild type cells are sensitive to combined

14 sorafenib and aspirin

15 We expanded our investigation to cell lines harboring KRASG12 mutations, investigating the

16 effects of combined sorafenib and aspirin treatment in the lung carcinoma cell line A549. The

17 dose response profile showed that A549 is insensitive to single agent sorafenib and aspirin

18 (Figure 1E), however the combination produced a dose-dependent response with an

19 effective sorafenib concentration as low as 500nM when combined with 2mM aspirin, and a

20 strong cytotoxic effect at 1µM sorafenib and 2mM aspirin (Figure 1F). We confirmed the

21 efficacy of the combination by MTT assay, which showed a significantly reduced cell

22 proliferation over 72h (Supplementary Figure 1F). The combination was also effective in the

23 KRASG12-mutant lung cancer cell line H358 (Supplementary Figure 1G) suggesting that

24 RAS-mutant cell lines in general are susceptible to this combination. Furthermore,

25 sorafenib/aspirin strongly reduced cell proliferation and viability in BRAF-mutant melanoma

26 cell lines WM164 and WM983B as measured by MTT (Figure 1G) and crystal violet staining

27 (Supplementary Figure 1H). By contrast, RAS/RAF wild type keratinocytes (HaCaT) or the

28 RAS/RAF-wild type breast cancer cell line SkBr3 only showed a minor response to combined

Hammerlindl et. al

1 sorafenib/aspirin (Figure 1H). Similarly the BRAF/NRAS-wild type melanoma cell line D24

2 was only moderately affected by the combination (Supplementary Figure 1I), suggesting

3 that the cytotoxic effects of sorafenib and aspirin are specific for RAS/RAF-mutant cancers.

4 The mutation status and sensitivity to combined sorafenib/aspirin of all tested cell lines is

5 summarized in Table 1.

7 The combination of sorafenib and aspirin activates ERK and AMPK pathways

8 Mutant RAS results in increased RAF/MEK/ERK signaling, while sorafenib inhibits BRAF and

9 CRAF amongst others, thereby inhibiting MAPK signaling (34). In addition to inhibiting

10 COX1/2, Aspirin has been shown to increase AMP-activated protein kinase (AMPK) signaling

11 (35). We therefore investigated these pathways in sorafenib/aspirin-exposed cells. Contrary

12 to our expectations, the combination treatment resulted in the increased activation of ERK1/2

13 (MAPK1/2) and the phosphorylation of the AMPK substrate acetyl-Coenzyme A carboxylase

14 (ACC) in NRAS- (Figure 2A), KRAS- (Figure 2B) and BRAF-mutant (Figure 2C and D)

15 cells. By contrast, RAS/RAF-wild type keratinocytes (HaCaT) (Figure 2E) and BRAF/NRAS-

16 wild type melanoma cells (D24) (Figure 2F), which are both less sensitive to the combination

17 of sorafenib and aspirin, showed no or only a subtle ERK and/or AMPK pathway activation,

18 suggesting that these pathways could be crucial for the cytotoxic effects of the combination

19 treatment. Sorafenib 5μM, which showed toxicity in WM1366 cells, also resulted in an

20 increase of pERK and pACC (Figure 2A), suggesting that single agent mediated toxicity

21 affects similar pathways in these cells. Furthermore, activation of the AMPK and MAPK

22 pathways was already observed after 4 hours of treatment (Supplementary Figure 2).

23 Because there is a significant lack of specific treatment options for RAS-mutant driven

24 cancers, and RAS- and RAF-mutant cancer cells showed the same pathway activation

25 pattern, we conducted all further experiments in NRAS- and KRAS-mutant cell lines. To test

26 for the contribution of both pathways, we blocked them using the selective mitogen-activated

27 protein kinase kinase (MEK) inhibitor trametinib and the AMPK inhibitor compound C

28 (dorsomorphin). While single treatment with the MEK inhibitor resulted in a dose-dependent

Hammerlindl et. al

1 increase in toxicity, the inhibition of MEK reduced sorafenib and aspirin induced toxicity in

2 NRAS- and KRAS-mutant cells (Figure 3A). Similarly, compound C showed a dose-

3 dependent toxicity while rescuing the cells if combined with sorafenib and aspirin (Figure 3B)

4 confirming the importance of activated AMPK and ERK signaling for the toxicity of the

5 combination treatment. Simultaneous inhibition of AMPK and MEK in cells treated with the

6 combination therapy increased cell viability over inhibition of either pathway alone

7 (Supplementary Figure 3A and C). The decreased toxicity of sorafenib and aspirin after

8 inhibition of MEK and/or AMPK was also confirmed by MTT assays (Supplementary Figure

9 3B and D), even though no additional benefit of combining trametinib and compound C was

10 detected using this assay. The observations of the crystal violet stainings suggest that AMPK

11 and ERK1/2 activation are independent. Indeed, we found that treatment with sorafenib,

12 aspirin and trametinib resulted in activated AMPK signaling but blocked ERK signaling,

13 whereas treatment with sorafenib, aspirin and compound C resulted in decreased AMPK

14 signaling but activated ERK1/2 (Figure 3C). This indicates that AMPK and ERK pathway

15 activation are independent events that synergistically mediate cytotoxicity of the combination

16 treatment. To confirm the specificity of the observations using pharmacological inhibitors, we

17 used sequence-specific shRNAs to silence BRAF and AMPKα1/2 (PRKAA1/PRKAA2) in

18 NRAS-mutant melanoma cells (WM1366). BRAF- or AMPKα1/2-silenced cells showed

19 decreased sensitivity to sorafenib and aspirin compared to empty vector transduced control

20 cells (Figure 3D and E) suggesting that both of these proteins are involved in

21 sorafenib/aspirin-mediated cytotoxicity. It has been shown that BRAF inhibitors can induce

22 paradoxical MAPK pathway activation by promoting BRAF/CRAF dimerization in a RAS-

23 dependent manner in RAS-mutant and RAS/RAF-wild type cancers (36). CRAF

24 immunoprecipitation showed that sorafenib in combination with aspirin, but not single agent

25 treatment, induced strong BRAF/CRAF complex formation in NRAS-mutant WM1366 (Figure

26 3F) and BRAF-mutant WM164 melanoma cells (Figure 3G). Interestingly, BRAF shows an

27 electrophoretic mobility shift in WM1366 which recently has been linked to phosphorylation

28 and increased activity of BRAF as part of a high molecular weight complex in RAS mutant

Hammerlindl et. al

1 cancer cells (37). We then tested the involvement of BRAF/CRAF complexes in the

2 activation of the ERK pathway by using the pan-RAF inhibitor LY3009120 that has been

3 shown to inhibit active dimers (38). Similar to trametinib, LY3009120 reduced the toxicity of

4 sorafenib and aspirin profoundly in both NRAS- and KRAS-mutant cells (Figure 3H),

5 suggesting that RAF activation is required for sorafenib- and aspirin-induced cytotoxicity.

6 Combining sorafenib, aspirin and LY3009120 significantly rescued cell proliferation as

7 determined by MTT assays confirming previous findings (Supplementary Figure 3E and F).

8 LY3009120 alone or in combination with aspirin resulted in moderate activation of ERK

9 signaling, which, even though observed at a higher concentration, is in line with previous

10 reports (38). The combination of LY3009120 with sorafenib and aspirin blocked

11 hyperactivation of ERK signaling without inhibiting AMPK pathway activity (Figure 3I), again

12 suggesting that MAPK and AMPK pathway activation are independent from each other.

13 Taken together, combined sorafenib and aspirin simultaneously hyperactivate ERK and

14 AMPK signaling, which both contribute to decreased cell viability in RAS-mutant cancers.

16 The combination of sorafenib with other AMPK activators shows no synergistic effects

17 As a proof of principle, we tested the combination of aspirin with the BRAFV600E specific drug

18 dabrafenib, which has been reported to paradoxically activate ERK in NRAS-mutant

19 melanoma (9,39). Combining dabrafenib at concentrations that are 100-1000 times higher

20 than usually used for melanoma (40), with aspirin resulted in a profound decrease of cell

21 viability in RAS-mutant cells (Figure 4A, Supplementary Figure 4A). Only the combination

22 induced hyperactivation of ERK and AMPK signaling whereas dabrafenib alone, which

23 showed no effect on cell viability, only resulted in hyperactivated ERK (Figure 4B). Similar to

24 dabrafenib in combination with aspirin, dabrafenib combined with the antidiabetic drug

25 metformin, which is known to be an AMPK activator, also exerted cytotoxic effects

26 (Supplementary Figure 4B) and activated ERK and AMPK signaling (Figure 4B). We then

27 tested whether sorafenib in combination with other AMPK activators is also effective.

28 Interestingly, the combination of sorafenib with metformin showed no synergistic effect in

Hammerlindl et. al

1 NRAS- and KRAS-mutant cells (Figure 4C, Supplementary Figure 4C). On the molecular

2 level combined sorafenib and metformin resulted in activation of the AMPK pathway but only

3 modest activation of ERK signaling (Figure 4D). We then hypothesized that the mechanistic

4 differences of AMPK activation of metformin and aspirin might influence the synergistic

5 effects. Metformin is an indirect AMPK activator as it inhibits oxidative phosphorylation (41)

6 while aspirin interacts directly with AMPK. To account for this mechanistic difference, we

7 combined sorafenib with the allosteric AMPK activator A-769662, which has been shown to

8 activate AMPK by interacting with the same protein region as aspirin (35). This combination

9 also failed to decrease cell viability in RAS-mutant cancers (Figure 4E). Like metformin, the

10 combination with A-769662 resulted in AMPK pathway activation while failing to trigger

11 hyperactivated ERK signaling (Figure 4F). Similar to the in vitro results, the combination of

12 sorafenib and aspirin showed a significant synergistic effect in NRAS- and KRAS-mutant

13 xenograft mouse models (Figure 5A and B). By comparison, the combination of sorafenib

14 and metformin failed to decrease tumor growth (Supplementary Figure 5A) confirming the

15 superior efficacy of the sorafenib and aspirin combination. Immunohistochemical analysis of

16 the A549-derived tumors showed that the majority of the sorafenib or sorafenib/aspirin-

17 treated tumors consisted of necrotic/dead cells with only the periphery of the tumors showing

18 p-ERK positivity (Supplementary Figure 5B-F). By applying an immunoreactivity score it

19 was found that sorafenib/aspirin-treated tumors displayed a higher staining intensity

20 compared to sorafenib-treated tumors (Figure 5C). Immunoblotting of A549-derived

21 xenografts tumors treated with three doses of combined sorafenib/aspirin or vehicle (n=3)

22 further showed significant activation of AMPK signaling, indicating a similar pathway

23 activation pattern as observed in vitro (Figure 5D). These data demonstrate that sorafenib in

24 combination with aspirin is a suitable strategy to target RAS-mutant cancers, whereas other

25 AMPK activators such as metformin or A-769662 show no synergistic anti-tumorigenic

26 effects in combination with sorafenib (Figure 5E).

28 Discussion 16

Hammerlindl et. al

1 The identification of the prominent melanoma driver mutation BRAFV600E was followed by the

2 development of clinically effective drugs specifically targeting this mutation. Despite the

3 emergence of resistance to these drugs, treatment significantly improves patient survival and

4 is viewed as a model for the development of oncogene-directed targeted therapies (42).

5 Intensive research efforts have been undertaken to develop drugs specifically targeting

6 mutant NRAS, the second-most common driver mutation of melanoma, but no agents have

7 been approved by the FDA to date (6,7). Besides the lack of effective targeted therapies,

8 mutant NRAS is also correlated with shorter survival after diagnosis of late stage disease

9 compared to NRAS/BRAF-wild type or BRAF-mutant melanoma (4), highlighting the

10 aggressive nature of melanomas driven by this mutation. Here we report the novel

11 combination of sorafenib and aspirin to target RAS-mutant cancers. Considering that the

12 clinically achievable sorafenib plasma concentration of approximately 5 µM is associated with

13 severe adverse effects (43), reducing the effective sorafenib dosage by combining it with

14 clinically achievable aspirin concentrations (44,45) could be a promising strategy to

15 overcome toxicity issues. However, adverse effects triggered by aspirin and strategies to

16 prevent these adverse effects must also be considered (46). The best characterized

17 mechanism of action of aspirin is the inhibition of the cyclooxygenase enzymes COX1 and

18 COX2, both of which are acetylated at a serine residue in the active site of the enzyme,

19 abolishing enzyme activity (47). Interestingly, the combination with other NSAIDs, SC-560,

20 ibuprofen or celecoxib which inhibit COX1 and/or COX2 (48,49), showed no synergistic

21 toxicity suggesting a mechanism independent of COX1/2 inhibition. While epidemiological

22 studies provide compelling evidence that NSAIDs are associated with reduced risk of cancer

23 (50), some reports also suggest that aspirin rather than other NSAIDs show a protective

24 effect in some cancer types (51). Indeed, several reports have suggested that the cancer

25 preventive action of the selective COX2 inhibitor celecoxib is the result of COX2-independent

26 effects (52,53). Mechanistically, inhibition of COX1/2 by aspirin is unlike other NSAIDs and

27 the result of a covalent irreversible modification (49). While acetylation of COX1 completely

28 blocks enzyme activity, acetylation of COX2 modifies the enzyme activity leading to the

Hammerlindl et. al

1 generation of lipoxins (54), which inhibit cell proliferation and angiogenesis of colorectal

2 cancer (55). While a contribution of such lipoxins to the observed toxicity of sorafenib and

3 aspirin cannot be excluded, the lack of synergy of sorafenib with other NSAIDs that target

4 COX1/2 strongly suggests that the effect of aspirin is independent of inhibition of

5 cyclooxygenase enzymes. Recently it has been shown that aspirin-derived salicylate

6 activates AMPK by directly binding to and allosterically inhibiting dephosphorylation of AMPK

7 (35). This aspect of aspirin is intriguing as activation of AMPK has been suggested to have

8 anti-tumorigenic effects in certain contexts (56), especially in melanoma (57). Accordingly,

9 constitutive activation of MAPK signaling by mutant BRAF has been shown to inhibit LKB1

10 via ERK and p90Rsk, inhibiting AMPK activation in response to energy stress and promoting

11 melanoma cell proliferation (58,59). Furthermore, the combination of AMPK activators, like

12 metformin or phenformin, with MAPK pathway inhibitors has been reported to be an effective

13 strategy to increase treatment response in BRAF-mutant melanoma (60). Thus, it was

14 surprising to see that neither metformin nor A-769662 showed synergistic effects in

15 combination with sorafenib. In our experiments, aspirin alone did not activate AMPK

16 signaling, which could be the direct result of LKB1-AMPK uncoupling. Sorafenib on the other

17 hand, has been reported to activate AMPK in an LKB1 and/or CAMKK2-dependent manner

18 (61), which, together with the aspirin-mediated stabilization of the active state of AMPK could

19 result in the strongly increased activation of the AMPK pathway. ShRNA-mediated silencing

20 of AMPK profoundly reduced the cytotoxic effects of sorafenib and aspirin co-treatment,

21 suggesting that AMPK activation is necessary but not sufficient to increase the cytotoxicity of

22 sorafenib. Inhibition of ERK signaling also reduced the cytotoxicity of the combination, and

23 the ability of the pan-RAF inhibitor LY3009120 and BRAF silencing to block the cytotoxicity of

24 the combination, demonstrates the importance of BRAF in the observed hyperactivation of

25 ERK signaling. Considering that both dominant melanoma driver mutations result in

26 constitutive activation of ERK signaling and that sorafenib is a multi-kinase inhibitor that is

27 supposed to inhibit the MAPK pathway (62), the involvement of hyperactivated MAPK

28 signaling for the cytotoxicity of the combination treatment was unexpected. However, it is

Hammerlindl et. al

1 well known that sorafenib induces the observed BRAF/CRAF dimerization in BRAF-wild type

2 cells, which can lead to paradoxical activation of the ERK signaling pathway (36,39,63). We

3 also observed sorafenib/aspirin but not sorafenib or aspirin alone, mediated BRAF/CRAF

4 dimerization in BRAF-mutant cells which has not been described for more specific BRAF

5 inhibitors like PLX4720 (36). It is noteworthy that unlike other ATP-competitive RAF

6 inhibitors, sorafenib binds and stabilizes RAF in a different conformation (64). It is possible

7 that this difference in the binding mode is important for the observed synergy between

8 sorafenib and aspirin and the resulting increased BRAF/CRAF dimerization in BRAF-mutant

9 cells. It is noteworthy that other mechanisms like dysregulation of the complex negative

10 feedback regulation of Dual-specificity phosphatases (DUSP) (65) could also be involved in

11 the hyperactivation of ERK signaling and therefore contribute to sorafenib/aspirin induced

12 anti-tumorigenic effects. While the detailed downstream effects of sorafenib/aspirin mediated

13 hyperactivation of ERK and AMPK signaling remain elusive, it has been recently shown that

14 hyperactivated ERK signaling induces apoptosis in B-Raf mutant cancers (66), resulting

15 inoncogene-induced senescence and negative selection of RAF and RAS double mutations

16 (67) or inhibit melanoma growth and induce autophagy in vitro and in vivo (68). Beside the

17 effectiveness of sorafenib in combination with aspirin to target NRAS-mutant melanoma,

18 KRAS-mutant lung adenocarcinoma cell lines also proved to be sensitive to this combination.

19 Sorafenib has recently been tested in a phase 2 clinical trial for the treatment of non-small

20 cell lung cancer (NSCLC) with KRAS mutations, which showed relevant but modest clinical

21 activity in patients (69). Our results suggest that the use of sorafenib in combination with

22 aspirin is a promising strategy for the treatment of KRAS-mutant NSCLC. Taken together, we

23 identified molecular drivers mediating the cytotoxic effects of sorafenib in combination with

24 aspirin. This work provides a strong rationale to repurpose two clinically approved drugs to

25 treat cancer types that lack specific therapeutic options.

26 27 Acknowledgments

Hammerlindl et. al

1 We thank Dr. Gerald Hoefler (Institute of Pathology, Medical University of Graz, Graz,

2 Austria) for kindly providing A549, H358 and HaCaT cells and Dr Fiona Simpson (The

3 University of Queensland Diamantina Institute, Brisbane, QLD) for kindly providing SkBr3

4 cells. The authors also acknowledge the help provided by the Translational Research

5 Institute (TRI) FACS, histology and microscopy core facilities.

7 References

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1 Table 1: Overview of sensitivity to combined sorafenib and aspirin Cell line Type Mutation status Sorafenib/Aspirin sensitivity WM1366 Melanoma NRAS Yes WM1361A Melanoma NRAS Yes WM852 Melanoma NRAS Yes CJM Melanoma NRAS Yes A549 Lung carcinoma KRAS Yes H358 Non-small cell lung KRAS Yes cancer WM164 Melanoma BRAF Yes WM983B Melanoma BRAF Yes HaCaT Immortalized RAS/RAF wild type No keratinocyte SkBr3 Breast cancer RAS/RAF wild type No D24 Melanoma RAS/RAF wild type No 2

Hammerlindl et. al

1 Figure 1: The combination of sorafenib and aspirin synergistically decreases cell

2 viability in RAS-mutant cancers

3 A, Crystal violet staining of WM1366 treated with increasing concentrations of sorafenib

4 (250nM-5µM) or increasing concentrations of aspirin (500 µM-2 mM) for 72 h. B, Crystal

5 violet staining of WM1366 treated with increasing concentrations of sorafenib (250 nM-5 µM)

6 in the presence of increasing concentrations of aspirin (500 µM-2 mM) for 72h. C, Drug

7 synergy assessed using the Bliss independence model. Green indicates synergistic, yellow

8 indicates additive and red indicates antagonistic effects of the drugs. D, Caspase 3 activation

9 following indicated drug exposures measured after 48h. *** indicates a P value ≤ 0.001, **

10 indicates P ≤ 0.01. E, Crystal violet staining of A549 treated with increasing concentrations of

11 sorafenib (250nM-5µM) or increasing concentrations of aspirin (500µM-2mM) for 72h. F,

12 Crystal violet staining of A549 exposed to increasing concentrations of sorafenib (250nM-

13 5µM) in the presence of increasing concentrations of aspirin (500µM-2mM) for 72h. G, Cell

14 proliferation of WM164 and WM983B BRAF-mutant melanoma cells following exposure to

15 sorafenib (1µM), aspirin (2mM) or the combination for 72h. *** indicates a P value ≤ 0.001. H,

16 Crystal violet staining of HaCaT or SkBr3 cells exposed to increasing concentrations of

17 sorafenib (250nM-5µM) alone or in combination with aspirin (2mM) for 72h.

18 19 20 Figure 2: Sorafenib and aspirin hyperactivate ERK and AMPK signaling in RAS-mutant

21 cells

22 Immunoblotting of whole cell lysates of WM1366 (A), A549 (B) WM164 (C), WM983B (D),

23 HaCat (E) and D24 (F) exposed to sorafenib (1μM), aspirin (2mM) or the combination for 48

24 h, using specific antibodies for phospho-Acetyl CoA Carboxylase (p-ACC), total Acetyl CoA

25 Carboxylase (t-ACC), phospho-ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2). β- Actin

26 was used as loading control. Immunoblots were quantified using ImageJ. The ratio of

27 phosphorylated to total protein normalized to control is shown above the respective blots.

Hammerlindl et. al

1 Figure 3: Inhibiting either AMPK or ERK activation reduces sorafenib/aspirin

2 sensitivity.

3 A, Crystal violet staining of WM1366 or A549 exposed to increasing concentrations of

4 trametinib (5nM-25 nM) and in the presence of sorafenib (1µM) and aspirin (2mM). B, Crystal

5 violet staining of WM1366 or A549 exposed to increasing concentrations of compound C

6 (1µM -10 µM) and in the presence of sorafenib (1µM) and aspirin (2mM). C, Immunoblotting

7 of whole cell lysates of WM1366 exposed to trametinib, compound C and/or sorafenib and

8 aspirin using specific antibodies for phospho-Acetyl CoA Carboxylase (p-ACC), total Acetyl

9 CoA Carboxylase (t-ACC), phospho-mTOR (p-mTOR), total mTOR (t-mTOR), phospho-

10 ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2). β- Actin was used as loading control. D,

11 Crystal violet staining of WM1366 transduced with shRNA targeting BRAF and treated with

12 sorafenib and/or aspirin for 72h. Knockdown was confirmed by immunoblotting. E, Crystal

13 violet staining of WM1366 transduced with shRNA targeting AMPK α1 and AMPK α2 and

14 treated with sorafenib and/or aspirin for 72h. Knockdown was confirmed by immunoblotting.

15 F and G, Immunoprecipitation using CRAF-specific antibodies after exposure to sorafenib

16 and/or aspirin at the indicated concentrations for 24h. Co-immunoprecipitation and total

17 BRAF expression was assessed by immunoblotting using specific antibodies for CRAF and

18 BRAF. β- Actin was used as loading control. H, Crystal violet staining of WM1366 or A549

19 exposed to increasing concentrations of the pan-RAF inhibitor LY3009120 (250nM-1µM) and

20 in the presence of sorafenib (1µM) and aspirin (2mM) for 72h. I, Immunoblotting of whole cell

21 lysates of WM1366 exposed to LY3009120 (1µM) and in combination with sorafenib (1µM)

22 and aspirin (2mM) for 48 hours using specific antibodies for phospho Acetyl CoA

23 Carboxylase (p-ACC), total Acetyl CoA Carboxylase (t-ACC), phospho-ERK1/2 (p-ERK1/2)

24 and total ERK1/2 (t-ERK1/2). β- Actin was used as loading control. Immunoblots were

25 quantified using ImageJ. The ratio of phosphorylated to total protein normalized to control is

26 shown above the respective blots.

Hammerlindl et. al

1 Figure 4: Activation of either AMPK or ERK signaling is not sufficient to induce

2 synergistic toxicity in combination with sorafenib.

3 A, C and E, Crystal violet staining of WM1366 or A549 exposed to the indicated drugs for

4 72h. B, D and F, Immunoblotting of whole cell lysates of WM1366 exposed to the indicated

5 drugs for 48 hours using specific antibodies for phospho-Acetyl CoA Carboxylase (p-ACC),

6 total Acetyl CoA Carboxylase (t-ACC), phospho-ERK1/2 (p-ERK1/2) and total ERK1/2 (t-

7 ERK1/2). Immunoblots were quantified using ImageJ. The ratio of phosphorylated to total

8 protein normalized to control is shown above the respective blots.

10 Figure 5: Sorafenib in combination with aspirin increases treatment efficacy in vivo

11 A and B, 1x106 WM1366 (A) or 2.5x106 A549 cells (B) were injected into

12 immunocompromised mice (n=4 mice per group). Mice were treated daily with the indicated

13 drugs. Statistical significance of results was calculated using an unpaired t-test. n.s. P > 0.05

14 ** P ≤ 0.01. C, Immunoreactivity score of phospho-ERK staining intensities of tumors

15 generated from A549 (Supplementary Figure 5 B-E) * P ≤ 0.05. D, Immunoblotting of A549-

16 derived xenografts following 54h of treatment with either vehicle or combined sorafenib

17 (15mg/kg) and aspirin (200mg/kg) using specific antibodies for phospho-Acetyl CoA

18 Carboxylase (p-ACC), total Acetyl CoA Carboxylase (t-ACC), phosphor-AMPK (p-AMPK),

19 total AMPK (t-AMPK), phospho-ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2).

20 Immunoblots were quantified using ImageJ. The ratio of phosphorylated to total protein

21 normalized to mean of control is shown above the respective blots (right panel) and plotted

22 as a graph (left panel). * indicates a P value ≤ 0.05. E, Proposed model of the synergistic

23 effects elicited by the combination of sorafenib and aspirin. The combination activates the

24 MAPK and AMPK pathways, which together induce cell death in RAS-mutant cancer.

Acetylsalicylic Acid Governs the Effect of Sorafenib in RAS- Mutant Cancers

Heinz Hammerlindl, Dinoop Ravindran Menon, Sabrina Hammerlindl, et al.

Clin Cancer Res Published OnlineFirst December 1, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-16-2118

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2017/10/28/1078-0432.CCR-16-2118.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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