Targeting NRAS-Mutant Cancers with the Selective STK19 ...€¦ · 3/10/2020 · Author Manuscript Published OnlineFirst on March 10, 2020; DOI: 10.1158/1078-0432.CCR-19-2604 . 6

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Targeting NRAS-Mutant Cancers with the Selective STK19 Kinase Inhibitor

Chelidonine

Ling Qian1,2, 4, Kun Chen1,2, 4, Changhong Wang3, Zhen Chen1,2, Zhiqiang Meng1,2, Peng Wang1,2

1 Department of Integrative Oncology, Fudan University Shanghai Cancer Center, 270 Dong An

Road, Shanghai 200032, China.

2 Department of Oncology, Shanghai Medical College, Fudan University, 130 Dong An Road,

Shanghai 200032, China.

3Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine,

Shanghai 201203, China.

4 These authors contributed equally to this work.

Running title: Chelidonine for NRAS-mutant cancer treatment.

Keywords: NRAS, STK19, cancer, Chelidonine

Financial support: This study was supported by the National Natural Science Foundation of

China (81622049, 81871989); the Shanghai Science and Technology Committee Program

(19XD1420900) and the Shanghai Education Commission Program (17SG04).

Corresponding author: Dr. Peng Wang, Department of Integrative Oncology, Fudan University

Shanghai Cancer Center, 270 Dong An Road, Shanghai 200032, China. Tel: 86-21-64175590-

83638; Fax: 86-21-64437657. E-mail: [email protected].

Research. on December 11, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on March 10, 2020; DOI: 10.1158/1078-0432.CCR-19-2604

http://clincancerres.aacrjournals.org/

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Disclosure of Potential Conflicts of Interest: The authors declare no potential conflicts of

interest.

Word count: 209 for abstract; 4582 for main text.

Total number of figures/tables: 6




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Translational Relevance

Oncogenic mutations in NRAS promote tumorigenesis, and novel anti-NRAS inhibitors are

urgently needed for cancer treatment. STK19 kinase was recently identified as a novel activator

of NRAS and a potential therapeutic target for NRAS-mutant melanomas. In this study, we

identified chelidonine, a natural compound, as a potent and selective inhibitor of STK19 kinase

activity. Chelidonine effectively inhibited proliferation and induced apoptosis in a panel of

cancer cells harboring NRAS mutations. Chelidonine also suppressed NRAS-driven tumor

growth in a mouse model while displaying minimal toxicity. These data indicate that chelidonine

can suppress the growth of NRAS-mutant cancer cells and could represent a novel option for the

treatment of such malignancies.




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ABSTRACT

Purpose: Oncogenic mutations in NRAS promote tumorigenesis. Although novel anti-NRAS

inhibitors are urgently needed for the treatment of cancer, the protein is generally considered

“undruggable” and no effective therapies have yet reached the clinic. STK19 kinase was recently

reported to be a novel activator of NRAS and a potential therapeutic target for NRAS-mutant

melanomas. Here, we describe a new pharmacological inhibitor of STK19 kinase for the

treatment of NRAS-mutant cancers.

Experimental Design: The STK19 kinase inhibitor was identified from a natural compound

library using a luminescent phosphorylation assay as the primary screen followed by verification

with an in vitro kinase assay and immunoblotting of treated cell extracts. The anti-tumor potency

of chelidonine was investigated in vitro and in vivo using a panel of NRAS-mutant and NRAS

wild-type cancer cells.

Results: Chelidonine was identified as a potent and selective inhibitor of STK19 kinase activity.

In vitro, chelidonine treatment inhibited NRAS signaling, leading to reduced cell proliferation

and induction of apoptosis in a panel of NRAS-mutant cancer cell lines, including melanoma,

liver, lung, and gastric cancer. In vivo, chelidonine suppressed the growth of NRAS-driven tumor

cells in nude mice while exhibiting minimal toxicity.

Conclusions: Chelidonine suppresses NRAS-mutant cancer cell growth and could have utility as

a new treatment for such malignancies.




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INTRODUCTION

The RAS family of small GTPases (HRAS, NRAS, and KRAS) are binary molecular switches

that transition between an active guanosine triphosphate (GTP)-bound state and an inactive

guanosine diphosphate (GDP)-bound state (1-4). Stimulation of many cell surface receptors

activates membrane-bound RAS proteins and its downstream signaling pathways, including

RAF–MEK–ERK and PI3K–AKT, which culminate in the promotion of cell growth and

suppression of cell death (5, 6). Aberrant RAS activity due to oncogenic mutations is frequently

associated with the promotion of tumorigenesis (7, 8); indeed, RAS mutations are present in 20–

30% of all human cancers (8, 9). Melanoma is characterized by gain-of-function hotspot

mutations in NRAS at glutamate 61 (Q61) (10, 11), including arginine, lysine, and leucine

mutations (Q61R, Q61K, and Q61L), which are present in approximately 30% of melanomas.

These mutations result in a constitutively GTP-bound active conformation of NRAS that drives

the malignant transformation of melanocytes (10-13). However, pharmacological targeting of

mutant NRAS proteins and the downstream signaling pathways has been challenging. Some

drugs with the potential to treat NRAS-mutant cancers have been developed, such as the MEK

inhibitor binimetinib, which showed some improvement in progression-free survival of patients

with NRAS-mutant melanoma in a phase III trial; however, binimetinib is still in clinical

development (14, 15).

Recent work demonstrated that the functionally uncharacterized serine/threonine kinase

STK19 is a novel activator of NRAS (16, 17). STK19 phosphorylates NRAS at the

evolutionarily conserved residue serine 89 (S89), which enhances binding between NRAS and its

effector proteins, activates downstream signaling pathways, and induces malignant

transformation of melanocytes (17). Crossing of NRAS Q61R transgenic mice with mice

harboring melanocyte-specific expression of STK19 or the gain-of-function mutant STK19

D89N enhances melanoma formation, confirming the ability of STK19 to stimulate NRAS

signaling (17, 18). These observations suggest that selective STK19 inhibitors could provide

urgently needed therapeutic options to suppress the growth of NRAS-mutant tumors.

Chelidonine is one of the most abundant bioactive isoquinoline alkaloids in extracts of

the plant Chelidonium majus, which is also known as the greater celandine (Papaveraceae) and is

widely distributed throughout Europe and Asia (19). Crude extracts of Chelidonium majus and




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purified chelidonine have both been shown to possess anti-tumor properties, including inhibition

of cell proliferation, potentiation of apoptosis, and suppression of cell migration and invasion, in

cell lines from such diverse cancers as uveal melanoma, head and neck cancer, gastric carcinoma,

liver cancer, and breast cancer (20-24). For example, chelidonine potentiates apoptosis in the

HCT116 (KRAS G13D) human colon cancer cell line by inhibiting the NF-κB signaling pathway

(25), and it suppresses the migration and invasion of MDA-MB-231 (KRAS G13D) human

breast cancer cells by inhibiting formation of the integrin-linked kinase–PINCH–α-parvin

complex (26). However, the precise mechanisms of action of chelidonine and its direct targets in

cancer cells remain unclear, greatly hindering its translation to the clinic.

In the present study, we screened a natural compound library using a phosphorylation

assay-based approach and identified chelidonine as a potent and selective inhibitor of STK19.

Using biochemical and cellular assays, we show that chelidonine is an ATP-competitive inhibitor

of STK19 activity and blocks proliferation and induces apoptosis in a panel of NRAS-mutant

cancer cell lines via inhibition of pathways downstream of NRAS, including RAF–MEK–ERK

and PI3K–AKT. Similarly, chelidonine impaired cancer cell growth in vivo while having

minimal toxicity. Our results suggest that pharmacological inhibition of STK19 by chelidonine

may provide a novel option for targeting NRAS-mutant cancers.




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MATERIALS AND METHODS

Cell lines

SK-MEL-2, SK-MEL-28, SK-MEL-31, HepG2, Hep3B, NCI-H446, SW-1271, HCT116, and

MDA-MB-231 cell lines were purchased from the American Type Culture Collection (Manassas,

VA, USA); WM2032, WM3406, and WM1366 cell lines were purchased from Rockland

Immunochemicals; and SNU-719 and SNU-216 cells were purchased from the Korean Cell Line

Bank (Seoul, Korea). The mutation status of these cell lines is as below: SK-MEL-2 (Q61R),

WM1366 (Q61L), WM2032 (Q61R), WM3406 (Q61K), HepG2 (Q61L), SW-1271 (Q61R),

SNU-719 (Q61L), Hep3B (NRAS-WT), NCI-H446 (NRAS-WT), SNU-216 (NRAS-WT),

HCT116(G13D), MDA-MB-231 (G13D), SK-MEL-28 (NRAS-WT and BRAFV600E), and SK-

MEL-31 (NRAS-WT and BRAFV600E). All cell lines were maintained in Dulbecco’s Modified

Eagle’s Medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL

streptomycin. Cell lines underwent routine testing for mycoplasma every 3 months (last

confirmed negative date, October 24, 2019). The genetic identity of the cell lines was confirmed

by short tandem repeat profiling. The cell lines were used for experiments within 10 passages

after thawing.

Clinical specimens

Twenty-eight tumor samples were collected from NRAS mutated melanoma patients after

surgical resection at Fudan University Shanghai Cancer Center (Shanghai, China) from January

2011 to May 2017. Written informed consent was obtained from all patients in accordance with

institutional guidelines before sample collection. The study was approved by the committees for

ethical review of research at Fudan University Shanghai Cancer Center.

Animal studies

All animal experiments were conducted in accordance with the guidelines of the National

Institutes of Health for the Care and Use of Laboratory Animals. The study protocol was also

approved by the Committee on the Use of Live Animals in Teaching and Research, Fudan

University, Shanghai, China. Mice were housed with a time-cycle of 12 h/12 h light/dark cycle




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(6:00 AM/PM). Mice were allowed free access to an irradiated diet and sterilized water. The

mice were monitored daily for signs related to their health status and distress.

For toxicity profiling of chelidonine, C57BL/6J mice were injected intraperitoneally (i.p.)

with vehicle (normal saline containing 5% [w/v] Kolliphor HS 15; Sigma) or chelidonine (10 or

20 mg/kg in vehicle) once daily and body weights were measured daily. After 21 days, the mice

were euthanized, and blood and organs were collected. Serum aspartate and alanine

aminotransferase (AST and ALT) activity was measured using assay kits (Abcam) according to

the manufacturer’s instructions. The organs were processed by fixing in 4% paraformaldehyde

and embedding in paraffin using standard protocols. Tissues were cut into 5-μm-thick sections,

stained with hematoxylin and eosin (H&E) and observed by light microscopy.

The pharmacokinetic profile of chelidonine was analyzed in mice injected i.p. with

chelidonine at 10 mg/kg. Chelidonine concentrations in mouse plasma were measured using an

ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)

method established for this study.

In vivo xenograft experiments were performed as described previously (27). Briefly,

2 × 106 SK-MEL-2 (NRAS Q61R), WM1366 (NRAS Q61L) or SK-MEL-28 (NRAS-wild-type

[WT]) cells were mixed with Matrigel (1:1) and injected subcutaneously into the left flanks of 8-

week-old female nude mice. Tumor size was measured every 3 days with calipers, and tumor

volumes were calculated using the following formula: length × width2 × 0.5. When the tumor

volume reached approximately 200 mm3, mice were injected with vehicle or chelidonine (10 or

20 mg/kg) i.p. once daily. On the indicated days, the mice were euthanized, and melanoma

xenografts were excised, weighed, and processed for further analysis.

Screening of STK19 kinase inhibitors

The optimal conditions for the 96-well Promega ADP-Glo® kinase assay (incubation time,

STK19 and ATP concentration) were previously determined according to the manufacturer’s

protocol (17) and found to be 12.5 nM STK19, 6.36 μM of ATP, and 15 min incubation.

Individual compounds from the natural compound library (TargetMol) were added to the wells at

a final concentration of 10 μM. STK19 kinase activity was quantified based on the luminescence




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signal detected with a Tecan Infinite® M1000 Microplate Reader. The screening results are

presented as the percent inhibition of STK19 kinase activity relative to control levels.

Compounds exhibiting ≥50% relative inhibition of STK19 activity in the primary screen were

selected for secondary evaluation.

Immunoblot analysis

Cells were lysed in a buffer containing 50 mM Tris pH 7.4, 150 mM NaCl, 0.5 mM EGTA,

0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 10% glycerol, and

complete protease inhibitor cocktail (Roche). The lysates were then homogenized and

centrifuged at 14,000 rpm at 4°C for 15 min. Protein concentrations were determined using a

Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Cell lysates were incubated with

PierceTM Lane Marker Reducing Sample Buffer at 100°C for 10 min, and proteins were

separated with 8–16% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad), and

transferred to PVDF membranes (Bio-Rad). After blocking, the membranes were incubated with

specific primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary

antibodies. The antibodies and suppliers were: monoclonal anti-β-actin (AC15), monoclonal anti-

Flag M2 (A8592), monoclonal anti-HA (H6533), HRP-conjugated anti-rabbit (A-4914), and

HRP-conjugated anti-mouse (A4416) antibodies (all from Sigma-Aldrich); and anti-

phosphorylated (p-) MEK1/2 (Ser217/221) (9121), anti-MEK (9122), anti-p-ERK1/2

(Thr202/Tyr204) (9101), anti-ERK1/2 (9102), anti-p-AKT (Ser473) (9271), anti-AKT (9272),

anti-cleaved caspase-3 (Asp175) (9661), anti-cleaved caspase-7 (Asp198) (9491), and anti-

cleaved poly (ADP-ribose) polymerase (PARP, Asp214) (9541) (all from Cell Signaling

Technology). A custom-generated antibody against p-NRAS (Ser89) was obtained from

Hangzhou Huaan Biotechnology Co. Ltd.

Immunohistochemistry (IHC)

A human tissue microarray (TMA) containing 28 melanoma tissues with NRAS mutation were

established. Unstained 3 um-thick sections were then prepared from paraffin-embedded tissues.

The sections were stained with primary antibodies at 4°C overnight. Staining with the secondary




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antibody and avidin-biotin peroxidase complex was performed according to the standard

protocols provided by the manufacturer (Vector Laboratories, Burlingame, CA, USA). All

procedures were performed by two independent assessors and one pathologist, none of whom

had any previous knowledge of the clinical outcomes of the cases. An immunoglobulin-negative

control was used to rule out nonspecific binding. The primary antibodies used were: anti-STK19

(251814) and anti- phosphorylated (p-) ERK1/2 (Thr202/Tyr204) (138482) antibodies (all from

Abcam); and anti-p-MEK1/2 (Ser217/221) (9121) and anti-p-AKT (Ser473) (9271) antibodies

(all from Cell Signaling Technolog). IHC intensities were assessed by a semiquantitative system

according to the immunoreactive score (IRS). The IRS is obtained by multiplying the staining

intensity by the percentage of positive cells, resulting in an IRS between 0 and 12. Briefly, the

staining intensity (SI) was categorized as 0 (negative), 1 (weak), 2 (intermediate), or 3 (strong),

and the percentage of positive cells (PP) was scored as 0 (0% positive), 1 (1%–25%), 2 (26%–

50%), 3 (51%–75%), or 4 (76%–100%). The IHC staining IRS = SI × PP. Two senior

pathologists performed the scorings independently in a blinded manner.

Quantitative reverse-transcription PCR (qRT-PCR)

Total RNA was extracted using a Qiagen RNeasy kit (Invitrogen) as previously described (28),

and cDNA was synthesized with SuperScript II Reverse Transcriptase (Invitrogen). Aliquots of

cDNA (30 ng) were amplified by qPCR with TaqManTM Gene Expression Master Mix (Thermo

Fisher Scientific). The mRNA levels of the genes of interest (PHLDA1, DUSP4, ETV4, and

SPRY2) were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA

in the same samples. The qPCR data were analyzed using the comparative CT method. All PCR

reactions were performed in triplicate.

In vitro kinase assay

The reaction mixture contained recombinant human HA-NRAS protein preloaded with GTP,

purified recombinant human STK19-Flag protein in kinase buffer (20 mM MnCl2, 50 mM

HEPES, pH 8.0), 300 µM AMP, and the indicated concentrations of ATP. Samples were

incubated for various times at 30°C. Proteins were then immunoprecipitated using anti-HA- or




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anti-Flag-conjugated beads, separated by SDS-PAGE, transferred to membranes, and subjected

to immunoblotting to detect p-NRAS.

Colony formation assays

Assays were performed as described previously (29). Briefly, melanoma cells were placed in 6-

well plates at a density of 2.5 × 103 cells/well and incubated with the indicated concentrations of

chelidonine. After 14 days, colonies were stained with 0.1% crystal violet, visualized by light

microscopy, and enumerated.

Cell viability assays

Cell viability was determined using a CyQUANT® NF Cell Proliferation Assay Kit (Invitrogen)

according to the manufacturer’s protocol. Briefly, cells were plated in 96-well microplates at 500

cells/well and incubated with the indicated concentrations of chelidonine for 4 days.

CyQUANT® NF dye solution was then added to the wells and fluorescence intensity was

measured with a fluorescence microplate reader using a 485/520 nm filter set. Cell viability is

presented as the fold-change relative to the initial cell number.

5-Ethynyl-2′-deoxyuridine (EdU) proliferation assay

EdU incorporation into DNA was detected using a Click-iT™ EdU Alexa Fluor™ 488 Flow

Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Briefly, melanoma cells (1 × 106 per sample) were harvested, washed twice in PBS/1% BSA, and

fixed in 100 μL Click-iT fixative. After incubation for 15 min at room temperature in the dark,

the cells were washed twice in 1× saponin-based permeabilization and wash reagent and

incubated with the Click-iT EdU reaction cocktail for 30 min. For the staining of cellular DNA,

cells were washed once in 1× saponin-based permeabilization and wash buffer and incubated

with DNA staining solution for 15 min at room temperature in the dark. Cells were then filtered

through a 200 μm mesh and analyzed using a BD FACSCalibur flow cytometer (BD

Biosciences).




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Differential scanning fluorimetry assay

The thermal denaturation of purified recombinant STK19 protein was determined using a Protein

Thermal ShiftTM Dye Kit (Thermal Fisher Scientific) as described previously (17). In brief, the

purified recombinant protein was diluted to a final concentration of 10 μM in 100 mM of Tris

buffer (pH 8.0). Aliquots of 20 μL of the protein sample were mixed with chelidonine (final

concentration 100 μM) and a heat gradient (25°C to 99°C) was then applied using a QuantStudio

12K Flex Real-Time PCR System. The melting curve was recorded and the melting temperature

was determined using the inflection points of the d(RFU)/dT plots.

Statistical analysis

All quantitative data are presented as the mean ± SD or SEM of at least three independent

experiments. Significant differences between groups were assessed using Student’s t-test.

Survival analysis was performed using the Kaplan–Meier method and compared using the log-

rank test. All analyses were performed using GraphPad Prism 7 or Microsoft Excel 2010. A p

value of 0.05 was considered statistically significant.




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RESULTS

Chelidonine is a potent ATP-competitive inhibitor of STK19

To identify pharmacological regulators of STK19 kinase activity, we screened ~1500 natural

compounds using a luminescent phosphorylation-based assay with purified recombinant human

NRAS protein as the substrate (Fig. 1A). We obtained 20 preliminary hits that inhibited STK19

activity by ≥50% at 10 μM in two independent experiments (Fig. 1B). As a secondary screen, the

primary hits were incubated with the human melanoma cell line SK-MEL-2 (NRAS Q61R), and

phosphorylation of NRAS at S89 was detected by immunoblotting (30) of cell extracts with an

anti-p-NRAS (S89) antibody (Fig. 1C). The specificity of the antibody for S89-phosphorylated

NRAS was validated by dot blot analysis using biotinylated peptides (Supplementary Fig. S1A).

From these assays, we selected the top candidates, including chelidonine, lycorine, jatrorrhizine,

fangchinoline, and daurisoline, and their inhibitory effects on STK19 were confirmed with an in

vitro kinase assay. Of note, chelidonine and jatrorrhizine are both benzophenanthridine alkaloids

with similar molecular structures (Fig. 1D and Supplementary Fig. S1B). Among the hits,

chelidonine was the most potent inhibitor of STK19 kinase activity (IC50 125.5 ± 19.3 nM) and

was selected for in-depth evaluation (Fig. 1E and Supplementary Fig. S1C).

The inhibitory activity of chelidonine against STK19 was further validated using an in

vitro kinase assay with purified recombinant human STK19 and NRAS Q61R proteins. These

experiments demonstrated that chelidonine inhibited the phosphorylation of NRAS in a

concentration- and time-dependent manner (Fig. 1F, G). Moreover, chelidonine appeared to be

an ATP-competitive inhibitor of STK19, as indicated by the increase in chelidonine IC50 value

as the ATP concentration in the reaction mixture was increased (Fig. 1H). Chelidonine showed a

melting temperature (Tm) shift at 6.49°C (Fig. 1I), indicative of a high-affinity interaction

between chelidonine and STK19.

We next evaluated the selectivity of chelidonine for STK19 by performing a

KINOMEscan assay, which screens a panel of 468 kinases using an in vitro ATP-site

competition binding assay (31). The KINOMEscan assay scores are reported as the percentage

relative to the negative control signal (DMSO), set to 100%. A selectivity score (S-Score) (35) of

0.05 was determined for chelidonine, which was calculated by dividing the number of kinases

tested with less than indicated chelidonine inhibited 25 of the 468 kinases tested to <35% of




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control kinase activity (Supplementary Fig. S2A and Supplementary Tab. S1). Among these,

six kinases were inhibited to <10% of control activity in the presence of chelidonine: EGFR

(L858R, T790M), ERN1, MAP3K13, MAPK9, MATK, and RPS6KA4. To validate these

findings, we performed in vitro STK19 kinase assays using the six purified recombinant kinases.

As shown in Fig. 1E and Supplementary Fig. S2B, chelidonine was a more potent inhibitor of

STK19 compared with EGFR (L858R, T790M), ERN1, MAP3K13, MAPK9, MATK, and

RPS6KA4. Because of the relatively similar IC50 values of chelidonine towards STK19, EGFR,

and MAPK9, we further overexpressed Flag-tagged STK19, EGFR or MAPK9 into SK-MEL-2

melanoma cells to explore whether overexpression of STK19, EGFR, or MAPK9 modulates the

effects of chelidonine on cell growth. We observed that only the overexpression of STK19

reduced the inhibitory effects of chelidonine on the growth of SK-MEL-2 cells, but not EGFR or

MAPK9 (Supplementary Figure S2C), confirming the specificity of chelidonine towards

STK19. Taken together, these results demonstrate that chelidonine is a potent and highly-

selective ATP-competitive inhibitor of STK19 kinase.

Chelidonine inhibits NRAS-mediated signaling

STK19-induced phosphorylation of NRAS S89 enhances NRAS activity and promotes

downstream signaling (17). We confirmed STK19 activity in human NRAS-mutant melanoma

tissues with immunohistochemical staining and observed a positive correlation between STK19

expression and activation of NRAS downstream MAPK and AKT signaling pathways

(Supplementary Fig. S3A, B). To determine whether chelidonine inhibits signaling downstream

of NRAS, we incubated chelidonine with a panel of human melanoma cell lines with various

NRAS mutations (SK-MEL-2 [NRAS Q61R], WM2032 [NRAS Q61R], WM3406 [Q61K], and

WM1366 [Q61L] (30, 32-34)), and examined NRAS pathway activation by immunoblotting.

Notably, chelidonine dose-dependently reduced the phosphorylation not only of endogenous

NRAS S89 but also of MEK, ERK1/2, and AKT in all four NRAS-mutant melanoma cell lines

(Fig. 2A). Consistent with these findings, qRT-PCR analysis indicated that chelidonine also

decreased the expression of the ERK transcriptional target genes PHLDA1, DUSP4, ETV4, and

SPRY2 (35) (Fig. 2B). Thus, chelidonine effectively inhibits activation of NRAS and its

downstream signaling pathways in melanoma cells.




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Chelidonine inhibits proliferation and induces apoptosis in NRAS-mutant tumor cells

Because oncogenic NRAS plays a critical role in promoting melanoma cell growth and

preventing cell death (1), we next evaluated these processes in SK-MEL-2, WM2032, WM3406,

and WM1366 melanoma cells after treatment with 5 or 20 μM chelidonine. Indeed, chelidonine

substantially inhibited the colony-forming ability (Fig. 3A, B), viability (Fig. 3C), and

proliferation (EdU incorporation) (Fig. 3D) of the cells. Furthermore, chelidonine induced

apoptosis of melanoma cells, as indicated by the appearance of the apoptosis effector proteins

cleaved caspase-3, caspase-7, and PARP (Fig. 3E, F) and by the increased activity of caspase

enzymes (Fig. 3F). To confirm that these effects of chelidonine were specific for STK19-

activated NRAS signaling pathways, we also explored its effect on two KRAS-mutant cancer

cells (HCT116 and MDA-MB-231) and two BRAF-mutant, NRAS-WT melanoma cells (SK-

MEL-28 and SK-MEL-31). Importantly, chelidonine did not significantly inhibit the growth of

either the KRAS-mutant cell lines (Supplementary Fig. S4) or the NRAS-WT melanoma cells

(Supplementary Fig. S5A, S5B). Collectively, these data indicate that chelidonine specifically

inhibits the proliferation and survival of NRAS-mutant melanoma cells.

Chelidonine exhibits minimal toxicity in mice

Next, we evaluated the clinical potential of chelidonine by determining its toxicity and

pharmacokinetic profile in mice. C57BL/6 mice were injected i.p. with 10 mg/kg chelidonine,

and blood was collected at various times thereafter. Plasma concentrations of chelidonine were

measured using an UHPLC-MS/MS method. The elimination half-life of chelidonine in mouse

plasma was 19.78 h (data not shown), which suggests that high concentrations of drug can be

maintained in the plasma by once daily injections. Next, we injected C57BL/6 mice i.p. with 0,

(vehicle), 10, or 20 mg/kg chelidonine once daily for 21 days and body weights were measured

daily. The mice displayed no overt clinical signs during this time and chelidonine treatment had

no significant effects on body weights (Fig. 4A). On day 21, the mice were euthanized, and

blood and tissues were collected for blood biochemistry and histopathological analyses,

respectively. Chelidonine had no significant apparent effects on hepatic function, as shown by

serum levels of AST and ALT (Fig. 4B), or on tissue integrity, as evaluated by histopathological




16

analysis of major organs (Fig. 4C). These results suggest that chelidonine has minimal toxicity in

vivo.

Chelidonine suppresses the growth of xenograft tumors harboring NRAS mutations

To confirm the in vivo therapeutic potential of chelidonine for the treatment of melanoma, we

injected SK-MEL-2 (NRAS Q61R) melanoma cells subcutaneously into nude mice and allowed

tumors to grow to approximately 200 mm3 in volume. Treatment was then initiated by once daily

i.p. injections with 0 (vehicle), 10 or 20 mg chelidonine. Chelidonine significantly and dose-

dependently reduced the volumes and weights of tumor xenografts compared with vehicle

treatment (Fig. 5A–C). Tumor-bearing mice treated with chelidonine also survived significantly

longer than mice treated with vehicle (Fig. 5D). Xenograft tissues were excised at the end of the

experiment and NRAS signaling pathway activation was assessed by immunoblotting of tumor

extracts. The results indicated that chelidonine inhibited NRAS signaling in a dose-dependent

manner, as illustrated by the reductions in phosphorylated NRAS S89, GTP-bound NRAS, and

phosphorylated MEK, ERK1/2, and AKT (Fig. 5E). The inhibitory effects of chelidonine on

xenograft tumor growth were also confirmed in another NRAS mutant WM1366 melanoma cells

(Supplementary Fig. S6A–S6C). In contrast, chelidonine did not suppress the growth of SK-

MEL-28 (NRAS-WT) melanoma cells (Supplementary Fig. S6D–S6F) confirming the

specificity of action of chelidonine observed in vitro. Taken together, these results provide

support for the in vivo therapeutic potential of chelidonine by demonstrating its ability to

specifically inhibit the growth of NRAS-mutant, but not NRAS-WT, melanoma.

Chelidonine inhibits the proliferation of various NRAS-mutant cancer cell lines

Our in vitro and in vivo studies thus far show that chelidonine is a potent inhibitor of NRAS-

mutant melanoma growth, with concomitant downregulation of NRAS, ERK, and AKT signaling.

To determine whether chelidonine can inhibit the progression of other types of NRAS-mutant

cancers, we examined its effects on the viability of HepG2 (NRAS Q61L), SW-1271 (NRAS

Q61R), and SNU-719 (NRAS Q61L) cell lines (Fig. 6A). Consistent with the inhibitory effects

of chelidonine on the growth of NRAS-mutant, but not NRAS-WT, melanoma cells and




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xenografts, the proliferation of these three cell lines was substantially inhibited compared with

vehicle (Fig. 6A). To confirm that these effects were mediated via inhibition NRAS and its

downstream signaling pathways, we examined phosphorylation of NRAS, MEK, ERK1/2, and

AKT in HepG2, SW-1271, and SNU-719 cells by immunoblotting. Indeed, phosphorylation of

each of these signaling proteins was inhibited by chelidonine treatment (Fig. 6B). In contrast,

similar experiments with the NRAS-WT counterparts of these cell lines (Hep3B, NCI-H446, and

SNU-216) revealed no significant effects of chelidonine on either cell proliferation or activation

of NRAS and downstream signaling proteins (Supplementary Fig. S7A and S7B), confirming

that chelidonine can inhibit the growth of various cancer cell lines harboring NRAS-mutant,

while NRAS-WT-expressing cells were unaffected. Taken together, these studies demonstrate

that chelidonine is a potent and selective STK19-targeting inhibitor that specifically blocks

oncogenic NRAS-driven tumor progression.




18

DISCUSSION

KRAS, HRAS, and NRAS (1, 3, 8, 9) were the first identified oncogenes, and it is now well

established that about 25% of all human cancers harbor activating mutations in at least one of

these proteins (8, 36). In particular, oncogenic KRAS mutations are present in 95% of pancreatic

ductal adenocarcinomas and 52% of colorectal adenocarcinomas, with the majority occurring at

G12. HRAS mutations (mainly G12 and Q61) are frequently associated with bladder cancer, and

20–30% of cutaneous melanomas are driven by NRAS Q61 mutations (36-38). Although

therapeutic modulation of RAS signaling has been a goal for decades, it has proven difficult to

develop strategies to directly inhibit RAS activity. Nevertheless, numerous alternative strategies

aimed at exploiting RAS-related vulnerabilities or targeting RAS regulators and effectors have

been studied.

RAS is activated at the plasma membrane following its prenylation by

farnesyltransferases (39, 40). Several farnesyltransferase inhibitors have been developed to block

this step (41); however, they have proven unsuccessful in clinical trials due to the alternative

modification of RAS by geranylgeranyl isoprenoid (42). G12C mutations in RAS create a pocket

for a potential covalent inhibitor, but this is a relatively minor RAS mutation in cancer (43).

Other important breakthroughs in anti-RAS therapies include the small molecule RAS-mimetic

rigosertib and pan-RAS ligands that block RAS binding to effector proteins containing a

common RAS-binding domain (44, 45). GTPase-activating proteins and guanine nucleotide

exchange factors (GEFs) are important regulators of the RAS activation/inactivation cycle (46),

and current efforts include therapeutic targeting of RAS/GEF interactions (47, 48). Synthetic

lethal strategies have also been employed to identify genes critical for the survival of NRAS-

mutant-expressing cancer cells but not those harboring NRAS-WT, and this approach has

identified STK33 (49), TBK1 (50), and PREX1 (51) as essential genes. RAS-mutant cancers are

highly dependent on upregulated metabolism to maintain their rapid growth (52), and targeting

of such metabolic dependence also represents a potentially promising route to therapy. Overall,

these new strategies to directly or indirectly target RAS and/or its key regulators and

vulnerabilities show great promise for the treatment of RAS-mutant-driven cancers.

Targeting of RAS post-translational modifications, particularly phosphorylation, is

another avenue to the development of anti-RAS therapies. Recent work identified STK19-




19

mediated phosphorylation of NRAS (S89) as a critical mechanism of mutant NRAS activation in

in melanocytes and their transformation into melanoma (17). STK19 is known to be a top driver

gene in this cancer (53), and somatic hotspot mutations in STK19 have been detected in about 5%

of melanomas (54) and 10% of skin basal cell carcinomas (55). However, the role of STK19 in

the regulation of RAS activity and tumor growth had not previously been appreciated. Here, we

observed a positive correlation between STK19 expression and activation of NRAS downstream

MAPK and AKT signaling pathways, and pharmacological inhibition of STK19 suppresses

activation of NRAS and the progression of NRAS-mutant-driven cancer both in vitro and in vivo,

substantiating the feasibility of STK19 as an anti-cancer therapeutic target.

Chelidonine has been reported to have broad pharmacological properties, including anti-

tumor, anti-inflammatory, anti-microbial, and anti-viral activities, but its mechanisms of action

and molecular functions were poorly understood (21-23, 25, 26). In this study, we demonstrated

that chelidonine directly binds to and inhibits STK19, thereby downregulating NRAS and its

downstream signaling pathways, leading to inhibition of tumor growth via suppression of

proliferation and induction of apoptosis. Chelidonine had good efficacy but minimal toxicity in

mice, and it also inhibited the growth of NRAS-mutant cancers of various origins, indicating that

this compound could form the basis for new therapies for multiple oncogenic RAS-driven

cancers.

The RAF–MEK–MAPK signaling cascade is the key RAS effector pathway for

promoting the proliferation and survival of RAS-mutant cancer cells (3). Numerous inhibitors of

this pathway have been demonstrated to improve clinical outcomes in patients with various

RAS- and RAF-mutant cancers (56-58). However, drug resistance invariably emerges in such

cancers, frequently involving an increase in oncogenic RAS/RAF driver mutations and

reactivation of the MEK–MAPK pathway (59-62). In response, various combination therapies

have been explored for the treatment of RAS-mutant cancers, such as the RAF inhibitor

sorafenib with aspirin (63), the MEK inhibitor trametinib with palbociclib, a CDK4/6 inhibitor

(64), and the BRAF/MEK inhibitors dabrafenib/trametinib with magnolol, a natural plant-

derived compound (65). These novel combinations have significantly improved the efficacy of

RAF–MEK–MAPK pathway inhibitors in RAS-mutant cancers. Considering that STK19 is an

activator of NRAS and thus acts upstream of the MEK–MAPK pathway, chelidonine might be a




20

useful additional therapy in combination with MEK inhibitors or other treatments for RAS-

mutant cancers. STK19 inhibition warrants further exploration in preclinical and clinical studies.




21

ACKNOWLEDGMENTS

We thank Dr. Shenglin Huang (Fudan University Shanghai Cancer Center) for technical support

and for valuable advice and discussions.




22

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26

FIGURE LEGENDS

Fig. 1. Chelidonine is a potent ATP-competitive inhibitor of STK19.

A, Screening strategy for the identification of STK19 kinase activity inhibitors.

B, Scatterplot of primary screening results. Twenty compounds that inhibited STK19 kinase

activity by >50% compared with control levels were identified and considered active hits (red

dots). Axes represent relative inhibition (percentage change in STK19 kinase activity relative to

that in the control group).

C, Immunoblotting of NRAS S89 phosphorylation in SK-MEL-2 melanoma cells following

treatment with the 20 screening hits for 12 h. Membranes were probed for p-S89-NRAS, NRAS,

and β-actin. Data shown are representative of three independent experiments.

D, Chemical structure of chelidonine.

E, In vitro kinase assay of chelidonine inhibition of STK19-mediated NRAS S89

phosphorylation. Data are the means ± SD relative to the control group (n = 3). IC50 represents

the median inhibitory concentration.

F, In vitro kinase assay of chelidonine inhibition of HA-NRAS Q61R (S89) phosphorylation (15

min incubation). Data shown are representative of three independent experiments.

G, In vitro kinase assay of the time and concentration dependence of chelidonine inhibition of

HA-NRAS Q61R (S89) phosphorylation (0–30 min, 0 or 10 µM chelidonine). Data shown are

representative of three independent experiments.

H, IC50 values of chelidonine for inhibition of STK19 kinase activity at ATP concentrations of

10, 30, 100, and 300 µM. Data are the means ± SD relative to control groups (n = 3).

I, Thermal shift assay of purified human recombinant STK19 protein (10 µM) in the presence of

chelidonine (100 µM).

Fig. 2. Chelidonine inhibits NRAS-mediated signaling.

A, SK-MEL-2, WM1366, WM2032, and WM3406 cells were treated with the indicated

concentrations of chelidonine and phosphorylation of NRAS (S89) and downstream signaling




27

molecules was detected by immunoblotting. Data shown are representative of three independent

experiments.

B, qRT-PCR analysis of ERK target gene transcription (PHLDA1, DUSP4, ETV4, and SPRY2)

in SK-MEL-2, WM1366, WM2032, and WM3406 cells treated with the indicated concentrations

of chelidonine. Error bars indicate 95% confidence intervals of triplicate measurements.

Fig. 3. Chelidonine inhibits cell proliferation and induces apoptosis in NRAS-mutant

melanoma cells.

A, SK-MEL-2, WM1366, WM2032, and WM3406 cells were treated with the indicated

concentrations of chelidonine and then seeded for colony formation assays. Data are the means ±

SD relative to the control group (n = 6) and are representative of three independent experiments.

B, Representative images of SK-MEL-2, WM1366, WM2032, and WM3406 cell colonies after

14 days of treatment with the indicated concentrations of chelidonine.

C, SK-MEL-2, WM1366, WM2032, and WM3406 cells were treated with the indicated

concentrations of chelidonine for 4 days and then seeded for viability assays. Data are the means

± SD relative to the control group (n = 6).

D, Representative results of EdU assay with SK-MEL-2, WM1366, WM2032, and WM3406

cells following treatment with 10 µM chelidonine for 24 h.

E, Immunoblotting of apoptosis markers (cleaved caspase-3 and -7 and cleaved PARP) in SK-

MEL-2, WM1366, WM2032, and WM3406 melanoma cells following treatment with

chelidonine for 4 days. Data shown are representative of three independent experiments.

F, SK-MEL-2, WM1366, WM2032, and WM3406 cells were treated with the indicated

concentrations of chelidonine for 4 days and caspase activity assays were performed. Data are

the means ± SD relative to the individual control group (n = 6).

Fig. 4. Chelidonine has minimal toxicity in mice.

A, Body weights of C57BL/6 mice injected i.p. with 0, 10, or 20 mg/kg chelidonine once daily

for 21 days. Data are the means ± SEM relative to control groups (n = 6).




28

B, AST and ALT activities in sera from C57BL/6 mice treated with 0, 10, or 20 mg/kg

chelidonine once daily for 21 days. Data are the means ± SD relative to the individual control

group (n = 3).

C, H&E staining of tissues from C57BL/6 mice treated with 0, 10, or 20 mg/kg chelidonine once

daily chelidonine for 21 days. Scale bar, 20 µm.

Fig. 5. Chelidonine suppresses the growth of NRAS-mutant melanoma xenografts.

A–C, Growth curves (A), tumor weights (B), and dissected tumors (D) from nude mice injected

subcutaneously with SK-MEL-2 cells (Q61R) and treated with 0, 10, or 20 mg/kg chelidonine

once daily. Volumes of visible tumors were measured every 3 days. Data are the means ± SEM

relative to the control group (n = 6).

D, Survival of SK-MEL-2 xenograft-bearing mice treated with 0, 10, or 20 mg/kg chelidonine

once daily. Results were compared using the log-rank test. p < 0.05 for 10 mg/kg chelidonine, p

< 0.001 for 20 mg/kg chelidonine vs control.

E, SK-MEL-2 xenograft tumors were collected for immunoblotting of phosphorylated NRAS

S89, GTP-bound active NRAS, and phosphorylated downstream signaling molecules. Data

shown are representative of three independent experiments.

Fig. 6. Chelidonine inhibits the proliferation of various NRAS-mutant cancer cells.

A, HepG2 (Q61L), SW-1271 (Q61R), and SNU-719 (Q61L) cells were treated with the indicated

concentrations of chelidonine and then seeded for cell viability assays. Data are the means ± SD

relative to the control group (n = 6).

B, Immunoblotting of phosphorylated NRAS (S89) and downstream signaling molecules in

HepG2, SW-1271, and SNU-719 cancer cells treated with the indicated concentrations of

chelidonine. Data shown are representative of three independent experiments.






















Published OnlineFirst March 10, 2020.Clin Cancer Res Ling Qian, Kun Chen, Changhong Wang, et al. Kinase Inhibitor ChelidonineTargeting NRAS-Mutant Cancers with the Selective STK19

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