Targeting NRAS-Mutant Cancers with the Selective STK19 Kinase

Published OnlineFirst March 10, 2020; DOI: 10.1158/1078-0432.CCR-19-2604

CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY

Targeting NRAS-Mutant Cancers with the Selective STK19 Kinase Inhibitor Chelidonine A C Ling Qian1,2, Kun Chen1,2, Changhong Wang3, Zhen Chen1,2, Zhiqiang Meng1,2, and Peng Wang1,2

ABSTRACT ◥ Purpose: Oncogenic mutations in NRAS promote tumori- cell extracts. The antitumor potency of chelidonine was investi- genesis. Although novel anti-NRAS inhibitors are urgently gated in vitro and in vivo using a panel of NRAS-mutant and needed for the treatment of cancer, the protein is generally NRAS wild-type cancer cells. considered “undruggable” and no effective therapies have yet Results: Chelidonine was identified as a potent and selective reached the clinic. STK19 kinase was recently reported to be a inhibitor of STK19 kinase activity. In vitro, chelidonine treatment novel activator of NRAS and a potential therapeutic target for inhibited NRAS signaling, leading to reduced cell proliferation NRAS-mutant melanomas. Here, we describe a new pharma- and induction of apoptosis in a panel of NRAS-mutant cancer cologic inhibitor of STK19 kinase for the treatment of NRAS- cell lines, including melanoma, liver, lung, and gastric cancer. mutant cancers. In vivo, chelidonine suppressed the growth of NRAS-driven Experimental Design: The STK19 kinase inhibitor was iden- tumor cells in nude mice while exhibiting minimal toxicity. tified from a natural compound library using a luminescent Conclusions: Chelidonine suppresses NRAS-mutant cancer phosphorylation assay as the primary screen followed by verifi- cell growth and could have utility as a new treatment for such cation with an in vitro kinase assay and immunoblotting of treated malignancies.

Introduction patients with NRAS-mutant melanoma in a phase III trial; however, binimetinib is still in clinical development (14, 15). The RAS family of small GTPases (HRAS, NRAS, and KRAS) are Recent work demonstrated that the functionally uncharacterized binary molecular switches that transition between an active GTP- serine/threonine kinase STK19 is a novel activator of NRAS (16, 17). bound state and an inactive GDP-bound state (1–4). Stimulation of STK19 phosphorylates NRAS at the evolutionarily conserved residue many cell surface receptors activates membrane-bound RAS proteins serine 89 (S89), which enhances binding between NRAS and its and its downstream signaling pathways, including RAF–MEK–ERK effector proteins, activates downstream signaling pathways, and and PI3K–AKT, which culminate in the promotion of cell growth induces malignant transformation of melanocytes (17). Crossing of and suppression of cell death (5, 6). Aberrant RAS activity due to NRAS Q61R transgenic mice with mice harboring melanocyte-specific oncogenic mutations is frequently associated with the promotion of expression of STK19 or the gain-of-function mutant STK19 D89N tumorigenesis (7, 8); indeed, RAS mutations are present in 20%–30% enhances melanoma formation, confirming the ability of STK19 to of all human cancers (8, 9). Melanoma is characterized by gain-of- stimulate NRAS signaling (17, 18). These observations suggest that function hotspot mutations in NRAS at glutamate 61 (Q61; refs. 10, 11), selective STK19 inhibitors could provide urgently needed therapeutic including arginine, lysine, and leucine mutations (Q61R, Q61K, options to suppress the growth of NRAS-mutant tumors. and Q61L), which are present in approximately 30% of melanomas. Chelidonine is one of the most abundant bioactive isoquinoline These mutations result in a constitutively GTP-bound active confor- alkaloids in extracts of the plant Chelidonium majus, which is also mation of NRAS that drives the malignant transformation of known as the greater celandine (Papaveraceae) and is widely distrib- melanocytes (10–13). However, pharmacologic targeting of mutant uted throughout Europe and Asia (19). Crude extracts of Chelidonium NRAS proteins and the downstream signaling pathways has been majus and purified chelidonine have both been shown to possess challenging. Some drugs with the potential to treat NRAS-mutant antitumor properties, including inhibition of cell proliferation, poten- cancers have been developed, such as the MEK inhibitor binimetinib, tiation of apoptosis, and suppression of cell migration and invasion, in which showed some improvement in progression-free survival of cell lines from such diverse cancers as uveal melanoma, head and neck cancer, gastric carcinoma, liver cancer, and breast cancer (20–24). For

1 example, chelidonine potentiates apoptosis in the HCT116 (KRAS Department of Integrative Oncology, Fudan University Shanghai Cancer Center, k Shanghai, China. 2Department of Oncology, Shanghai Medical College, Fudan G13D) human colon cancer cell line by inhibiting the NF- B signaling University, Shanghai, China. 3Institute of Chinese Materia Medica, Shanghai pathway (25), and it suppresses the migration and invasion of MDA- University of Traditional Chinese Medicine, Shanghai, China. MB-231 (KRAS G13D) human breast cancer cells by inhibiting – –a Note: Supplementary data for this article are available at Clinical Cancer formation of the integrin-linked kinase PINCH -parvin com- Research Online (http://clincancerres.aacrjournals.org/). plex (26). However, the precise mechanisms of action of chelidonine L. Qian and K. Chen contributed equally to this article. and its direct targets in cancer cells remain unclear, greatly hindering its translation to the clinic. Corresponding Author: Peng Wang, Fudan University Shanghai Cancer Center, In this study, we screened a natural compound library using a 270 Dong An Road, Shanghai 200032, China. Phone: 8621-6417-5590; Fax: 8621- – ﬁ 6443-7657; E-mail: [email protected] phosphorylation assay based approach and identi ed chelidonine as a potent and selective inhibitor of STK19. Using biochemical and Clin Cancer Res 2020;26:3408–19 cellular assays, we show that chelidonine is an ATP-competitive doi: 10.1158/1078-0432.CCR-19-2604 inhibitor of STK19 activity and blocks proliferation and induces 2020 American Association for Cancer Research. apoptosis in a panel of NRAS-mutant cancer cell lines via inhibition

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irradiated diet and sterilized water. The mice were monitored daily for Translational Relevance signs related to their health status and distress. Oncogenic mutations in NRAS promote tumorigenesis, and For toxicity profiling of chelidonine, C57BL/6J mice were injected novel anti-NRAS inhibitors are urgently needed for cancer treat- intraperitoneally with vehicle [normal saline containing 5% (w/v) ment. STK19 kinase was recently identified as a novel activator of Kolliphor HS 15; Sigma] or chelidonine (10 or 20 mg/kg in vehicle) NRAS and a potential therapeutic target for NRAS-mutant mel- once daily and body weights were measured daily. After 21 days, the anomas. In this study, we identified chelidonine, a natural com- mice were euthanized, and blood and organs were collected. Serum pound, as a potent and selective inhibitor of STK19 kinase activity. aspartate and alanine aminotransferase (AST and ALT) activity was Chelidonine effectively inhibited proliferation and induced apo- measured using Assay Kits (Abcam) according to the manufacturer's ptosis in a panel of cancer cells harboring NRAS mutations. instructions. The organs were processed by fixing in 4% paraformal- Chelidonine also suppressed NRAS-driven tumor growth in a dehyde and embedding in paraffin using standard protocols. Tissues mouse model while displaying minimal toxicity. These data indi- were cut into 5-mm thick sections, stained with hematoxylin and eosin cate that chelidonine can suppress the growth of NRAS-mutant (H&E), and observed by light microscopy. cancer cells and could represent a novel option for the treatment of The pharmacokinetic profile of chelidonine was analyzed in mice such malignancies. injected intraperitoneally with chelidonine at 10 mg/kg. Chelidonine concentrations in mouse plasma were measured using an ultra-high performance LC/MS-MS (UHPLC/MS-MS) method established for this study. of pathways downstream of NRAS, including RAF–MEK–ERK and In vivo xenograft experiments were performed as described previ- PI3K–AKT. Similarly, chelidonine impaired cancer cell growth in vivo ously (27). Briefly, 2 106 SK-MEL-2 (NRAS Q61R), WM1366 while having minimal toxicity. Our results suggest that pharmacologic (NRAS Q61L), or SK-MEL-28 (NRAS-WT) cells were mixed with inhibition of STK19 by chelidonine may provide a novel option for Matrigel (1:1) and injected subcutaneously into the left flanks of targeting NRAS-mutant cancers. 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 Materials and Methods approximately 200 mm3, mice were injected with vehicle or chelido- Cell lines nine (10 or 20 mg/kg, i.p.) once daily. On the indicated days, the mice SK-MEL-2, SK-MEL-28, SK-MEL-31, HepG2, Hep3B, NCI-H446, were euthanized, and melanoma xenografts were excised, weighed, and SW-1271, HCT116, and MDA-MB-231 cell lines were purchased from processed for further analysis. ATCC; WM2032, WM3406, and WM1366 cell lines were purchased from Rockland Immunochemicals; and SNU-719 and SNU-216 cells Screening of STK19 kinase inhibitors were purchased from the Korean Cell Line Bank (Seoul, Korea). The The optimal conditions for the 96-well Promega ADP-Glo Kinase mutation status of these cell lines is as below: SK-MEL-2 (Q61R), Assay (incubation time, and STK19 and ATP concentration) were WM1366 (Q61L), WM2032 (Q61R), WM3406 (Q61K), HepG2 previously determined according to the manufacturer's protocol (17) (Q61L), SW-1271 (Q61R), SNU-719 (Q61L), Hep3B [NRAS wild- and found to be 12.5 nmol/L STK19, 6.36 mmol/L of ATP, and 15 type (WT)], NCI-H446 (NRAS-WT), SNU-216 (NRAS-WT), minutes incubation. Individual compounds from the natural com- HCT116 (G13D), MDA-MB-231 (G13D), SK-MEL-28 (NRAS-WT pound library (TargetMol) were added to the wells at a final concen- and BRAFV600E), and SK-MEL-31 (NRAS-WT and BRAFV600E). tration of 10 mmol/L. STK19 kinase activity was quantified on the basis All cell lines were maintained in DMEM containing 10% FBS, 100 U/ of the luminescence signal detected with a Tecan Infinite M1000 mL penicillin, and 100 mg/mL streptomycin. Cell lines underwent Microplate Reader. The screening results are presented as the percent routine testing for Mycoplasma every 3 months (last confirmed inhibition of STK19 kinase activity relative to control levels. Com- negative date, October 24, 2019). The genetic identity of the cell lines pounds exhibiting ≥50% relative inhibition of STK19 activity in the was confirmed by short tandem repeat profiling. The cell lines were primary screen were selected for secondary evaluation. used for experiments within 10 passages after thawing. Immunoblot analysis Clinical specimens Cells were lysed in a buffer containing 50 mmol/L Tris, pH 7.4, Twenty-eight tumor samples were collected from patients with 150 mmol/L NaCl, 0.5 mmol/L EGTA, 0.5 mmol/L EDTA, 1 mmol/L NRAS-mutated melanoma after surgical resection at Fudan University phenylmethylsulfonyl fluoride, 1% Triton X-100, 10% glycerol, and Shanghai Cancer Center (Shanghai, China) from January 2011 to May complete Protease Inhibitor Cocktail (Roche). The lysates were then 2017. Written informed consent was obtained from all patients in homogenized and centrifuged at 14,000 rpm at 4C for 15 minutes. accordance with institutional guidelines before sample collection. The Protein concentrations were determined using a Pierce BCA Protein study was approved by the committees for ethical review of research at Assay Kit (Thermo Fisher Scientific). Cell lysates were incubated with Fudan University Shanghai Cancer Center (Shanghai, China). Pierce Lane Marker Reducing Sample Buffer at 100C for 10 minutes, and proteins were separated with 8%–16% Mini-PROTEAN TGX Animal studies Precast Protein Gels (Bio-Rad), and transferred to polyvinylidene All animal experiments were conducted in accordance with the difluoride membranes (Bio-Rad). After blocking, the membranes were guidelines of the NIH for the Care and Use of Laboratory Animals. The incubated with specific primary antibodies followed by horseradish study protocol was also approved by the Committee on the Use of Live peroxidase (HRP)-conjugated secondary antibodies. The antibodies Animals in Teaching and Research, Fudan University (Shanghai, and suppliers were: monoclonal anti-b-actin (AC15), monoclonal China). Mice were housed with a time-cycle of 12 hours/12 hours anti-Flag M2 (A8592), monoclonal anti-HA (H6533), HRP- light/dark cycle (6:00 am/pm). Mice were allowed free access to an conjugated anti-rabbit (A-4914), and HRP-conjugated anti-mouse

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(A4416) antibodies (all from Sigma-Aldrich); and anti- Flag protein in kinase buffer (20 mmol/L MnCl2, 50 mmol/L HEPES, phosphorylated (p-) MEK1/2 (Ser217/221) (9121), anti-MEK pH 8.0), 300 mmol/L AMP, and the indicated concentrations of ATP. (9122), anti-p-ERK1/2 (Thr202/Tyr204) (9101), anti-ERK1/2 Samples were incubated for various times at 30C. Proteins were then (9102), anti-p-AKT (Ser473) (9271), anti-AKT (9272), anti-cleaved immunoprecipitated using anti-HA- or anti-Flag–conjugated beads, caspase-3 (Asp175) (9661), anti-cleaved caspase-7 (Asp198) (9491), separated by SDS-PAGE, transferred to membranes, and subjected to and anti-cleaved poly (ADP-ribose) polymerase (PARP, Asp214) immunoblotting to detect p-NRAS. (9541) (all from Cell Signaling Technology). A custom-generated antibody against p-NRAS (Ser89) was obtained from Hangzhou Colony formation assays Huaan Biotechnology Co. Ltd. Assays were performed as described previously (29). Briefly, melanoma cells were placed in 6-well plates at a density of 2.5 103 cells/ IHC well and incubated with the indicated concentrations of chelidonine. A human tissue microarray containing 28 melanoma tissues with After 14 days, colonies were stained with 0.1% crystal violet, visualized NRAS mutation were established. Unstained 3-mm thick sections were by light microscopy, and enumerated. then prepared from paraffin-embedded tissues. The sections were stained with primary antibodies at 4C overnight. Staining with the Cell viability assays secondary antibody and avidin-biotin peroxidase complex was per- Cell viability was determined using a CyQUANT NF Cell Prolif- formed according to the standard protocols provided by the manu- eration Assay Kit (Invitrogen) according to the manufacturer's pro- facturer (Vector Laboratories). All procedures were performed by two tocol. Briefly, cells were plated in 96-well microplates at 500 cells/well independent assessors and one pathologist, none of whom had any and incubated with the indicated concentrations of chelidonine for previous knowledge of the clinical outcomes of the cases. An immu- 4 days. CyQUANT NF dye solution was then added to the wells and noglobulin-negative control was used to rule out nonspecific binding. fluorescence intensity was measured with a fluorescence microplate The primary antibodies used were: anti-STK19 (251814) and anti- reader using a 485/520 nm filter set. Cell viability is presented as the phosphorylated (p-) ERK1/2 (Thr202/Tyr204) (138482) antibodies fold-change relative to the initial cell number. (all from Abcam); and anti-p-MEK1/2 (Ser217/221) (9121) and anti-p-AKT (Ser473) (9271) antibodies (all from Cell Signaling 5-Ethynyl-20-deoxyuridine proliferation assay Technology). IHC intensities were assessed by a semiquantitative 5-Ethynyl-20-deoxyuridine (EdU) incorporation into DNA was system according to the immunoreactive score (IRS). The IRS is detected using a Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay obtained by multiplying the staining intensity by the percentage of Kit (Thermo Fisher Scientific) according to the manufacturer's instruc- positive cells, resulting in an IRS between 0 and 12. Briefly, the tions. Briefly, melanoma cells (1 106 per sample) were harvested, staining intensity (SI) was categorized as 0 (negative), 1 (weak), 2 washed twice in PBS/1% BSA, and fixed in 100 mL Click-iT fixative. (intermediate), or 3 (strong), and the percentage of positive cells After incubation for 15 minutes at room temperature in the dark, the (PP) was scored as 0 (0% positive), 1 (1%–25%), 2 (26%–50%), 3 cells were washed twice in 1 saponin-based permeabilization and (51%–75%), or 4 (76%–100%). The IHC staining IRS ¼ SI PP. wash reagent and incubated with the Click-iT EdU reaction cocktail for Two senior pathologists performed the scorings independently in a 30 minutes. For the staining of cellular DNA, cells were washed once in blinded manner. 1 saponin-based permeabilization and wash buffer and incubated with DNA staining solution for 15 minutes at room temperature in the Quantitative reverse-transcription PCR dark. Cells were then filtered through a 200-mm mesh and analyzed Total RNA was extracted using a Qiagen RNeasy Kit (Invitrogen) as using a BD FACSCalibur Flow Cytometer (BD Biosciences). described previously (28), and cDNA was synthesized with Super- Script II Reverse Transcriptase (Invitrogen). Aliquots of cDNA (30 ng) Differential scanning fluorimetry assay were amplified by qPCR with TaqManTM Gene Expression Master The thermal denaturation of purified recombinant STK19 protein Mix (Thermo Fisher Scientific). The mRNA levels of the genes of was determined using a Protein Thermal Shift Dye Kit (Thermal Fisher interest (PHLDA1, DUSP4, ETV4, and SPRY2) were normalized Scientific) as described previously (17). In brief, the purified recom- against GAPDH mRNA in the same samples. The qPCR data were binant protein was diluted to a final concentration of 10 mmol/L in 100 analyzed using the comparative Ct method. All PCR reactions were mmol/L of Tris buffer (pH 8.0). Aliquots of 20 mL of the protein sample performed in triplicate. were mixed with chelidonine (final concentration 100 mmol/L) and a heat gradient (25C–99C) was then applied using a QuantStudio 12K In vitro kinase assay Flex Real-Time PCR System. The melting curve was recorded and the The reaction mixture contained recombinant human HA-NRAS melting temperature was determined using the inflection points of the protein preloaded with GTP, purified recombinant human STK19- d(RFU)/dT plots.

Figure 1. Chelidonine (cheli) 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 hours. Membranes were probed for p-S89-NRAS, NRAS, and b-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 ¼ 3). IC50 represents the median inhibitory concentration. F, In vitro kinase assay of chelidonine inhibition of HA-NRAS Q61R (S89) phosphorylation (15-minute 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 minutes, 0 or 10 mmol/L 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 mmol/L. Data are the means SD relative to control groups (n ¼ 3). I, Thermal shift assay of puriﬁed human recombinant STK19 protein (10 mmol/L) in the presence of chelidonine (100 mmol/L).

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Figure 2. Chelidonine (Cheli) 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 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. , P < 0.001.

Statistical analysis nant human NRAS protein as the substrate (Fig. 1A). We obtained All quantitative data are presented as the mean SD or SEM of at 20 preliminary hits that inhibited STK19 activity by ≥50% at 10 least three independent experiments. Significant differences between mmol/L in two independent experiments (Fig. 1B). As a secondary groups were assessed using Student t test. Survival analysis was screen, the primary hits were incubated with the human melanoma performed using the Kaplan–Meier method and compared using the cell line SK-MEL-2 (NRAS Q61R), and phosphorylation of NRAS at log-rank test. All analyses were performed using GraphPad Prism 7 or S89 was detected by immunoblotting (30) of cell extracts with an Microsoft Excel 2010. A P value of 0.05 was considered statistically anti-p-NRAS (S89) antibody (Fig. 1C). The specificity of the significant. antibody for S89-phosphorylatedNRASwasvalidatedbydotblot analysis using biotinylated peptides (Supplementary Fig. S1A). From these assays, we selected the top candidates, including che- Results lidonine, lycorine, jatrorrhizine, fangchinoline, and daurisoline, and Chelidonine is a potent ATP-competitive inhibitor of STK19 their inhibitory effects on STK19 were confirmed with an in vitro To identify pharmacologic regulators of STK19 kinase activity, kinase assay. Of note, chelidonine and jatrorrhizine are both we screened approximately 1,500 natural compounds using a benzophenanthridine alkaloids with similar molecular structures luminescent phosphorylation-based assay with purified recombi- (Fig. 1D; Supplementary Fig. S1B). Among the hits, chelidonine

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was the most potent inhibitor of STK19 kinase activity (IC50 125.5 ATP concentration in the reaction mixture was increased (Fig. 1H). 19.3 nmol/L) and was selected for in-depth evaluation (Fig. 1E; Chelidonine showed a melting temperature (Tm) shift at 6.49 C Supplementary Fig. S1C). (Fig. 1I), indicative of a high-affinity interaction between chelidonine The inhibitory activity of chelidonine against STK19 was further and STK19. validated using an in vitro kinase assay with purified recombinant We next evaluated the selectivity of chelidonine for STK19 by human STK19 and NRAS Q61R proteins. These experiments dem- performing a KINOMEscan assay, which screens a panel of 468 kinases onstrated that chelidonine inhibited the phosphorylation of NRAS in a using an in vitro ATP site competition binding assay (31). The concentration- and time-dependent manner (Fig. 1F and G). More- KINOMEscan assay scores are reported as the percentage relative to over, chelidonine appeared to be an ATP-competitive inhibitor of the negative control signal (DMSO), set to 100%. A selectivity score STK19, as indicated by the increase in chelidonine IC50 value as the (35%) was defined as the percentage of total kinases (among the 468

Figure 3. Chelidonine (Cheli) 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 ¼ 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 ¼ 6). D, Representative results of EdU assay with SK-MEL-2, WM1366, WM2032, and WM3406 cells following treatment with 10 mmol/L chelidonine for 24 hours. E, Immunoblotting of apoptosis markers [cleaved caspase-3 (casp) 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 ¼ 6). , P < 0.001.

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kinases) whose kinase activity in the presence of chelidonine was findings, quantitative reverse-transcription PCR (qRT-PCR) analysis decreased to less than 35% of the control group (Supplementary indicated that chelidonine also decreased the expression of the ERK Fig. S2A; Supplementary Table S1). Among these, six kinases were transcriptional target genes PHLDA1, DUSP4, ETV4, and SPRY2 inhibited to <10% of control activity in the presence of chelidonine: (ref. 32; Fig. 2B). Thus, chelidonine effectively inhibits activation of EGFR (L858R and T790M), ERN1, MAP3K13, MAPK9, MATK, and NRAS and its downstream signaling pathways in melanoma cells. RPS6KA4. To validate these findings, we performed in vitro STK19 kinase assays using the six purified recombinant kinases. As shown Chelidonine inhibits proliferation and induces apoptosis in in Fig. 1E and Supplementary Fig. S2B, chelidonine was a more potent NRAS-mutant tumor cells inhibitor of STK19 compared with EGFR (L858R and T790M), ERN1, Because oncogenic NRAS plays a critical role in promoting MAP3K13, MAPK9, MATK, and RPS6KA4. Because of the relatively melanoma cell growth and preventing cell death (1), we next similar IC50 values of chelidonine toward STK19, EGFR, and MAPK9, evaluated these processes in SK-MEL-2, WM2032, WM3406, and we further overexpressed Flag-tagged STK19, EGFR, or MAPK9 into WM1366 melanoma cells after treatment with 5 or 20 mmol/L SK-MEL-2 melanoma cells to explore whether overexpression of chelidonine. Indeed, chelidonine substantially inhibited the colony- STK19, EGFR, or MAPK9 modulates the effects of chelidonine on forming ability (Fig. 3A and B), viability (Fig. 3C), and prolifer- cell growth. We observed that only the overexpression of STK19 ation (EdU incorporation; Fig. 3D) of the cells. Furthermore, reduced the inhibitory effects of chelidonine on the growth of SK- chelidonine induced apoptosis of melanoma cells, as indicated by MEL-2 cells, but not EGFR or MAPK9 (Supplementary Fig. S2C), theappearanceoftheapoptosiseffector proteins cleaved caspase-3, confirming the specificity of chelidonine toward STK19. Taken togeth- caspase-7, and PARP (Fig. 3E and F) and by the increased activity er, these results demonstrate that chelidonine is a potent and highly of caspase enzymes (Fig. 3F). To confirm that these effects of selective ATP-competitive inhibitor of STK19 kinase. chelidonine were specific for STK19-activated NRAS signaling pathways, we also explored its effect on two KRAS-mutant cancer Chelidonine inhibits NRAS-mediated signaling cells (HCT116 and MDA-MB-231) and two BRAF-mutant, NRAS- STK19-induced phosphorylation of NRAS S89 enhances NRAS WT melanoma cells (SK-MEL-28 and SK-MEL-31). Importantly, activity and promotes downstream signaling (17). We confirmed chelidonine did not significantly inhibit the growth of either the STK19 activity in human NRAS-mutant melanoma tissues with IHC KRAS-mutant cell lines (Supplementary Fig. S4) or the NRAS-WT staining and observed a positive correlation between STK19 expression melanoma cells (Supplementary Fig. S5A and S5B). Collectively, and activation of NRAS downstream MAPK and AKT signaling these data indicate that chelidonine specifically inhibits the prolif- pathways (Supplementary Fig. S3A and S3B). To determine whether eration and survival of NRAS-mutant melanoma cells. chelidonine inhibits signaling downstream of NRAS, we incubated chelidonine with a panel of human melanoma cell lines with various Chelidonine exhibits minimal toxicity in mice NRAS mutations [SK-MEL-2 (NRAS Q61R), WM2032 (NRAS Q61R), Next, we evaluated the clinical potential of chelidonine by deter- WM3406 (Q61K), and WM1366 (Q61L); refs. 30, 32–34], and exam- mining its toxicity and pharmacokinetic profile in mice. C57BL/6 mice ined NRAS pathway activation by immunoblotting. Notably, cheli- were injected intraperitoneally with 10 mg/kg chelidonine, and blood donine dose dependently reduced the phosphorylation not only of was collected at various times thereafter. Plasma concentrations of endogenous NRAS S89 but also of MEK, ERK1/2, and AKT in all four chelidonine were measured using an UHPLC/MS-MS method. The NRAS-mutant melanoma cell lines (Fig. 2A). Consistent with these elimination half-life of chelidonine in mouse plasma was

Figure 4. Chelidonine (Cheli) has minimal toxicity in mice. A, Body weights of C57BL/6 mice injected intraperitoneally with 0, 10, or 20 mg/kg chelidonine once daily for 21 days. Data are the means SEM relative to control groups (n ¼ 6). 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 ¼ 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 mm. n.s., not signiﬁcant.

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19.78 hours (data not shown), which suggests that high concentra- (Fig. 4C). These results suggest that chelidonine has minimal tions of drug can be maintained in the plasma by once daily toxicity in vivo. injections. Next, we injected C57BL/6 mice intraperitoneally with 0, (vehicle), 10, or 20 mg/kg chelidonine once daily for 21 days and Chelidonine suppresses the growth of xenograft tumors body weights were measured daily. The mice displayed no overt harboring NRAS mutations clinical signs during this time and chelidonine treatment had no To confirm the in vivo therapeutic potential of chelidonine for the significant effects on body weights (Fig. 4A). On day 21, the mice treatment of melanoma, we injected SK-MEL-2 (NRAS Q61R) mel- were euthanized, and blood and tissues were collected for blood anoma cells subcutaneously into nude mice and allowed tumors to biochemistry and histopathologic analyses, respectively. Chelido- grow to approximately 200 mm3 in volume. Treatment was then nine had no significant apparent effects on hepatic function, as initiated by once daily intraperitoneal injections with 0 (vehicle), shown by serum levels of AST and ALT (Fig. 4B), or on tissue 10, or 20 mg chelidonine. Chelidonine significantly and dose depen- integrity, as evaluated by histopathologic analysis of major organs dently reduced the volumes and weights of tumor xenografts

Figure 5. Chelidonine (Cheli) suppresses the growth of NRAS-mutant melanoma xenografts. Growth curves (A), tumor weights (B), and dissected tumors (C) 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 ¼ 6). , P < 0.05; , P < 0.01; , P < 0.001. 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 versus 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.

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compared with vehicle treatment (Fig. 5A–C). Tumor-bearing mice of action of chelidonine observed in vitro. Taken together, these results treated with chelidonine also survived significantly longer than mice provide support for the in vivo therapeutic potential of chelidonine by treated with vehicle (Fig. 5D). Xenograft tissues were excised at the end demonstrating its ability to specifically inhibit the growth of NRAS- of the experiment and NRAS signaling pathway activation was mutant, but not NRAS-WT, melanoma. assessed by immunoblotting of tumor extracts. The results indicated that chelidonine inhibited NRAS signaling in a dose-dependent man- Chelidonine inhibits the proliferation of various NRAS-mutant ner, as illustrated by the reductions in phosphorylated NRAS S89, cancer cell lines GTP-bound NRAS, and phosphorylated MEK, ERK1/2, and AKT Our in vitro and in vivo studies thus far show that chelidonine is a (Fig. 5E). The inhibitory effects of chelidonine on xenograft tumor potent inhibitor of NRAS-mutant melanoma growth, with concom- growth were also confirmed in another NRAS-mutant WM1366 itant downregulation of NRAS, ERK, and AKT signaling. To deter- melanoma cells (Supplementary Fig. S6A–S6C). In contrast, chelido- mine whether chelidonine can inhibit the progression of other types of nine did not suppress the growth of SK-MEL-28 (NRAS-WT) mel- NRAS-mutant cancers, we examined its effects on the viability of anoma cells (Supplementary Fig. S6D–S6F) confirming the specificity HepG2 (NRAS Q61L), SW-1271 (NRAS Q61R), and SNU-719 (NRAS

Figure 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 ¼ 6). , P < 0.001. 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.

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Chelidonine for NRAS-Mutant Cancer Treatment

Q61L) cell lines (Fig. 6A). Consistent with the inhibitory effects of therapies. Recent work identified STK19-mediated phosphorylation of chelidonine on the growth of NRAS-mutant, but not NRAS-WT, NRAS (S89) as a critical mechanism of mutant NRAS activation in melanoma cells and xenografts, the proliferation of these three cell melanocytes and their transformation into melanoma (17). STK19 is lines was substantially inhibited compared with vehicle (Fig. 6A). To known to be a top driver gene in this cancer (53), and somatic hotspot confirm that these effects were mediated via inhibiting NRAS and its mutations in STK19 have been detected in about 5% of melanomas (54) downstream signaling pathways, we examined phosphorylation of and 10% of skin basal cell carcinomas (55). However, the role of STK19 NRAS, MEK, ERK1/2, and AKT in HepG2, SW-1271, and SNU-719 in the regulation of RAS activity and tumor growth had not previously cells by immunoblotting. Indeed, phosphorylation of each of these been appreciated. Here, we observed a positive correlation between signaling proteins was inhibited by chelidonine treatment (Fig. 6B). In STK19 expression and activation of NRAS downstream MAPK and contrast, similar experiments with the NRAS-WT counterparts of AKT signaling pathways, and pharmacologic inhibition of STK19 these cell lines (Hep3B, NCI-H446, and SNU-216) revealed no sig- suppresses activation of NRAS and the progression of NRAS-mutant– nificant effects of chelidonine on either cell proliferation or activation driven cancer both in vitro and in vivo, substantiating the feasibility of of NRAS and downstream signaling proteins (Supplementary Fig. S7A STK19 as an anticancer therapeutic target. and S7B), confirming that chelidonine can inhibit the growth of Chelidonine has been reported to have broad pharmacologic prop- various cancer cell lines harboring NRAS mutant, while NRAS- erties, including antitumor, anti-inflammatory, antimicrobial, and WT–expressing cells were unaffected. Taken together, these studies antiviral activities, but its mechanisms of action and molecular func- demonstrate that chelidonine is a potent and selective STK19-targeting tions were poorly understood (21–23, 25, 26). In this study, we inhibitor that specifically blocks oncogenic NRAS-driven tumor demonstrated that chelidonine directly binds to and inhibits STK19, progression. 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 Discussion efficacy but minimal toxicity in mice, and it also inhibited the growth KRAS, HRAS, and NRAS (1, 3, 8, 9) were the first identified of NRAS-mutant cancers of various origins, indicating that this oncogenes, and it is now well established that about 25% of all human compound could form the basis for new therapies for multiple cancers harbor activating mutations in at least one of these pro- oncogenic RAS-driven cancers. teins (8, 36). In particular, oncogenic KRAS mutations are present The RAF–MEK–MAPK signaling cascade is the key RAS in 95% of pancreatic ductal adenocarcinomas and 52% of colorectal effector pathway for promoting the proliferation and survival of adenocarcinomas, with the majority occurring at G12. HRAS muta- RAS-mutant cancer cells (3). Numerous inhibitors of this pathway tions (mainly G12 and Q61) are frequently associated with bladder have been demonstrated to improve clinical outcomes in patients cancer, and 20%–30% of cutaneous melanomas are driven by NRAS with various RAS- and RAF-mutant cancers (56–58). However, Q61 mutations (36–38). Although therapeutic modulation of RAS drug resistance invariably emerges in such cancers, frequently signaling has been a goal for decades, it has proven difficult to develop involving an increase in oncogenic RAS/RAF-driver mutations strategies to directly inhibit RAS activity. Nevertheless, numerous and reactivation of the MEK–MAPK pathway (59–62). In alternative strategies aimed at exploiting RAS-related vulnerabilities response, various combination therapies have been explored for or targeting RAS regulators and effectors have been studied. the treatment of RAS-mutant cancers, such as the RAF inhibitor RAS is activated at the plasma membrane following its prenyla- sorafenib with aspirin (63), the MEK inhibitor trametinib with tion by farnesyltransferases (39, 40). Several farnesyltransferase palbociclib, a CDK4/6 inhibitor (64), and the BRAF/MEK inhibi- inhibitors have been developed to block this step (41); however, tors dabrafenib/trametinib with magnolol, a natural plant-derived they have proven unsuccessful in clinical trials due to the alternative compound (65). These novel combinations have significantly modification of RAS by geranylgeranyl isoprenoid (42). G12C improved the efficacy of RAF–MEK–MAPK pathway inhibitors mutations in RAS create a pocket for a potential covalent inhibitor, in RAS-mutant cancers. Considering that STK19 is an activator of but this is a relatively minor RAS mutation in cancer (43). Other NRAS and thus acts upstream of the MEK–MAPK pathway, important breakthroughs in anti-RAS therapies include the small- chelidonine might be a useful additional therapy in combination molecule RAS-mimetic rigosertib and pan-RAS ligands that block with MEK inhibitors or other treatments for RAS-mutant cancers. RAS binding to effector proteins containing a common RAS- STK19 inhibition warrants further exploration in preclinical and binding domain (44, 45). GTPase-activating proteins and guanine clinical studies. nucleotide exchange factors (GEF) are important regulators of the RAS activation/inactivation cycle (46), and current efforts include Disclosure of Potential Conflicts of Interest therapeutic targeting of RAS/GEF interactions (47, 48). Synthetic No potential conflicts of interest were disclosed. lethal strategies have also been employed to identify genes critical – for the survival of NRAS-mutant expressing cancer cells but not Authors’ Contributions those harboring NRAS-WT, and this approach has identified STK33 TBK1 PREX1 Conception and design: Z. Chen, Z. Meng, P. Wang (49), (50), and (51) as essential genes. RAS- Development of methodology: C. Wang, P. Wang mutant cancers are highly dependent on upregulated metabolism to Acquisition of data (provided animals, acquired and managed patients, provided maintain their rapid growth (52), and targeting of such metabolic facilities, etc.): L. Qian, K. Chen, C. Wang, P. Wang dependence also represents a potentially promising route to ther- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, apy. Overall, these new strategies to directly or indirectly target RAS computational analysis): L. Qian, K. Chen, C. Wang, P. Wang and/or its key regulators and vulnerabilities show great promise for Writing, review, and/or revision of the manuscript: L. Qian, K. Chen, C. Wang, – P. Wang the treatment of RAS-mutant driven cancers. Administrative, technical, or material support (i.e., reporting or organizing data, Targeting of RAS posttranslational modifications, particularly constructing databases): Z. Meng, P. Wang phosphorylation, is another avenue to the development of anti-RAS Study supervision: P. Wang

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Acknowledgments The costs of publication of this article were defrayed in part by the payment of page advertisement The authors thank Dr. Shenglin Huang (Fudan University Shanghai Cancer charges. This article must therefore be hereby marked in accordance Center, Shanghai, China) for technical support and for valuable advice and with 18 U.S.C. Section 1734 solely to indicate this fact. discussions. This study was supported by the National Natural Science Founda- tion of China (81622049 and 81871989), the Shanghai Science and Technology Committee Program (19XD1420900), and the Shanghai Education Commission Received August 8, 2019; revised December 2, 2019; accepted March 5, 2020; ﬁ Program (17SG04). published rst March 10, 2020.

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Targeting NRAS-Mutant Cancers with the Selective STK19 Kinase Inhibitor Chelidonine

Ling Qian, Kun Chen, Changhong Wang, et al.

Clin Cancer Res 2020;26:3408-3419. Published OnlineFirst March 10, 2020.

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