Triptolide Induces Cell Killing in Multidrug- Resistant Tumor Cells

Published OnlineFirst March 29, 2016; DOI: 10.1158/1535-7163.MCT-15-0753

Small Molecule Therapeutics Molecular Cancer Therapeutics Triptolide Induces Cell Killing in Multidrug- Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44 Jun-Mei Yi1, Xia-Juan Huan1, Shan-Shan Song1, Hu Zhou2, Ying-Qing Wang1, and Ze-Hong Miao1

Abstract

Multidrug resistance (MDR) is a major cause of tumor treat- induced by 72-hour treatment of triptolide, which may be due ment failure; therefore, drugs that can avoid this outcome are to partial rescue of RPB1 degradation. We suggest that a precise urgently needed. We studied triptolide, which directly kills MDR phosphorylation site on RPB1 (Ser1878) was phosphorylated by tumor cells with a high potency and a broad spectrum of cell CDK7 in response to triptolide. In addition, XPB and p44, two death. Triptolide did not inhibit P-glycoprotein (P-gp) drug efﬂux transcription factor TFIIH subunits, did not contribute to tripto- and reduced P-gp and MDR1 mRNA resulting from transcription lide-driven RPB1 degradation and cell killing, although XPB was inhibition. Transcription factors including c-MYC, SOX-2, OCT- reported to covalently bind to triptolide. Several clinical trials are 4, and NANOG were not correlated with triptolide-induced cell underway to test triptolide and its analogues for treating cancer killing, but RPB1, the largest subunit of RNA polymerase II, was and other diseases, so our data may help expand potential clinical critical in mediating triptolide's inhibition of MDR cells. Tripto- uses of triptolide, as well as offer a compound that overcomes lide elicited antitumor and anti-MDR activity through a universal tumor MDR. Future investigations into the primary molecular mechanism: by activating CDK7 by phosphorylating Thr170 in target(s) of triptolide responsible for RPB1 degradation may both parental and MDR cell lines and in SK-OV-3 cells. The suggest novel anti-MDR target(s) for therapeutic development. CDK7-selective inhibitor BS-181 partially rescued cell killing Mol Cancer Ther; 15(7); 1495–503. 2016 AACR.

Introduction overcoming activities of natural products were associated with the regulation of certain transcription factors. However, these Drug resistance, especially multidrug resistance (MDR), is a compounds do not hold promise for chemotherapeutic use, as major cause of tumor treatment failure (1). Clinically, few drugs they have relatively poor anticancer activity either in vitro are available that do not produce resistance, and resistance- (methyl spongoate and tanshinone I), in vivo (pseudolaric acid reversal agents are not available because most tested compounds B, YCH337, and MT series), or in clinical trials (salvicine). reverse MDR by inhibiting drug transporter function, such as Triptolide (Fig. 1A), a principle ingredient of Tripterygium P-glycoprotein (P-gp), which mediates tumor MDR. However, wilfordii Hook F, is a unique transcription inhibitor (13) with drug transporters are critical for physiologic processes at the potent anticancer activity. Its analogue minnelide is in clinical blood brain barrier, intestine, kidney, and liver (2). trials for cancer therapy, and several others are also in We reported that specific compounds can directly kill MDR clinical development, such as LLDT8 for rheumatoid arthritis and tumor cells without affecting the function of P-gp, such as the PG490-88 and WilGraf for graft rejection after organ transplan- natural products salvicine (3, 4), pseudolaric acid B (5, 6), methyl tation (14). Triptolide has been reported to covalently bind to the spongoate (7), tanshinone I (2, 8), and synthetic small molecules Cys342 residue of XPB, one subunit of TFIIH, a general transcrip- YCH337 (9) and MT series (10–12). Among them, the MDR- tion factor that regulates RNA polymerase I and II (RNAPII; refs. 15–17). Our previous work indicates that CDK7 and p44, two subunits of TFIIH, may contribute to the degradation of 1Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of RPB1 (the largest subunit of RNAPII) and therefore to tumor Sciences, Shanghai, P.R. China. 2CAS Key Laboratory of Receptor cell killing (13), but these relationships require clarification. Research, Shanghai Institute of Materia Medica, Chinese Academy of Here, we report that triptolide directly kills various tumor MDR Sciences, Shanghai, P.R. China. cells without inhibiting P-gp drug-efflux function. Reduced P-gp Note: Supplementary data for this article are available at Molecular Cancer by triptolide was due to transcription inhibition. Transcription Therapeutics Online (http://mct.aacrjournals.org/). factors including c-MYC, SOX-2, OCT-4, and NANOG do not Corresponding Authors: Ze-Hong Miao, Shanghai Institute of Materia Medica, contribute to proliferative inhibition of triptolide, but RPB1 Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, does facilitate this in MDR sublines and in parental tumor Shanghai 201203, China. Phone: 8621-5080-6820; Fax: 8621-5080-6820; E-mail: cell lines. We report that triptolide leads to the phosphorylation [email protected]; and Ying-Qing Wang, [email protected] of CDK7 at its Thr170 and RPB1 at its Ser1878. XPB and p44 doi: 10.1158/1535-7163.MCT-15-0753 appear not to be correlated with triptolide-driven RPB1 degrada- 2016 American Association for Cancer Research. tion and cell killing.

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Materials and Methods pended in PBS for 90 minutes and assessed with ﬂow cytometry with a FACSCalibur cytometer (BD Biosciences; ref. 2). Drugs, chemicals, and reagents Triptolide was purchased from Sigma-Aldrich, and BS-181 was from Selleck. Verapamil, doxorubicin (DX), and vincristine (VCR) Plasmid transfection were obtained from Melonepharma. RIPA lysis buffer, Protein Plasmid-expressing pMSCV-c-MYC (#18775) was obtained AþG beads, and Rhodamine 123 (Rh123) were from the Beyo- from Addgene. Transfection was conducted as reported previously time Institute of Biotechnology (Haimen, China). All antibodies in the published literature (18). were commercially available: RPB1 [carboxy-terminal domain (CTD) repeat], p-S5-RPB1, XPB, OCT-4, SOX-2, NANOG, ubiqui- RNA interference tin, and p-Thr170-CDK7 were from Abcam, GAPDH and IgG were XPB gene expression was reduced with speciﬁc siRNA duplexes from Beyotime Institute of Biotechnology (Haimen, China), from Santa Cruz Biotechnology. siRNA transfection was per- CDK7 and p44 were from Santa Cruz Biotechnology, and c-MYC formed with RNAiMAX Transfection Reagent (Invitrogen) accord- was from BD Biosciences. ing to the manufacturer's instructions.

Cell culture qRT-PCR Human cancer KB, IM-9, and MES/SA cell lines and doxoru- Total RNA was prepared with the TRIzol reagent (Invitrogen) bicin-selected resistant MES-SA/DX5 cell line were purchased and reverse transcribed into cDNA with a PrimeScript RT Reagent from the ATCC. Human cancer K562 cells and adriamycin-select- Kit (TaKaRa). cDNA was amplified with the SYBR Premix EX TaqII ed resistant K562/A02 cells were purchased from the Institute of Kit (TaKaRa) in a 7500 Fast Real-Time PCR System (Applied Hematology, Chinese Academy of Medical Sciences (Tianjin, Biosystems). The PCR program was as follows: 95 C, 30 seconds; China). Human cancer SK-OV-3 cells were obtained from the 40 cycles (for each cycle 95 C, 5 seconds; 64 C, 20 seconds; 72 C, Japanese Foundation of Cancer Research (Tokyo, Japan). 15 seconds); 72 C, 10 minutes. All primers were synthesized by GM21071 and GM02252 cells were purchased from the Coriell 0 0 Sangon as follows: 5 -GTATTCAACTATCCCACCC-3 (forward) Institute (Camden, NJ). A vincristine-selected resistant KB/VCR 0 0 0 and 5 - GCTTTATTTCTTTGCCATC-3 (reverse) for MDR1;5- subline was from the Sun Yat-Sen University of Medical Sciences 0 0 TCTACAATGAGCT GCGTGTG-3 (forward) and 5 -GGTGAG- (Guangzhou, China). During this study, all cell lines were authen- 0 0 GATCTTCAT-GAGGT-3 (reverse) for b-actin;5-CGTCTCCACA- ticated using the short tandem repeat (STR) profiling at Shanghai 0 0 CATCAGCACAA-3 (forward) and 5 -TGTTGGCAGC AGGA- Genesky Bio-Tech CO., LTD (KB and KB/VCR, May 2013; MES/SA 0 TAGTCCTT-3 (reverse) for c-MYC. and MES-SA/DX5, June 2013; SK-OV-3, August 2013; K562, March 2013; K562/A02 and IM-9, February 2014). Cells were – also periodically authenticated with morphologic inspection and Eliminating p44 expression with transcription activator like tested for mycoplasma contamination. Cell lines were cultured effector nuclease technique – according to the manufacturer's instructions. The transcription activator like effector nuclease technique (TALEN) technique is a new method for genome editing and fi Proliferative inhibition assays genetic modi cations by inducing DNA double-strand breaks IC values of different agents in adherent and suspended that stimulate error-prone nonhomologous end joining or 50 fi cells were measured using a sulforhodamine B (SRB; Sigma) homology-directed repair at speci c genomic locations (19). assay and the Cell Counting Kit-8 (Dojindo Laboratories) To eliminate p44 expression, TALEN was performed with a fi assay, respectively. Cells were seeded into 96-well plates, cul- FASTALE TALEN Kit (SiDanSai Biotechnology) speci cally tar- tured overnight, and treated with gradient concentrations of the geting p44. Transfected positive TALEN plasmids into SK-OV-3 m tested agents for 72 hours. Optical density for both assays was cells and screened with 1.5 g/mL puromycin. After puromycin read with a SpectraMax 190 (Molecular Devices). Averaged IC screening, surviving cells were cultured and selected for p44- 50 fi values were calculated using logit method from three indepen- de cient monoclonal cells. dent experiments (9). Immunoprecipitation Colony formation assays SK-OV-3 cells were treated with 1 mmol/L triptolide for the indi- KB and KB/VCR cells were plated into 6-well plates (200 cells/ cated times, and cells were treated as described previously (10). well). After overnight incubation, cells were treated with triptolide fi at the indicated concentrations for 72 hours and then xed with Determination of the RPB1 phosphorylation site induced by 10% trichloroacetic acid (TCA; Sangon), stained with 0.4% SRB, triptolide washed with 1% acetic acid, dried, and photographed. An RPB1-interacting protein mixture from immunoprecipitation was separated with SDS-PAGE separation, and in-gel Western blot analysis digestion was performed as reported previously (20). Then, Western blot analysis was performed as described previously in protein bands were excised, dehydrated with acetonitrile, and the published literature (10). digested with trypsin at 37C overnight. The resulting tryptic peptides were dissolved with 0.1% formic acid and centrifuged Rh123 efflux assays at 12,000 rpm for 15 minutes. Supernatant was analyzed by Cells were treated with 5 mmol/L verapamil or 1 mmol/L LC/MS-MS using an Orbitrap Elite high-resolution mass spec- triptolide for 90 minutes, followed by incubation with Rh123 trometer. MS-MS spectra were searched against the human (1 mg/mL) for 30 minutes. Then, cells were collected and sus- database using pFind software (21).

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Statistical analyses and concentration-dependent manners in parental cell lines All data, if applicable, were expressed as mean SD from at as reported previously (13) and in respective MDR sublines least three independent experiments. Comparisons between two (SupplementaryFig.S1AandS1BandFig.1D).However,short- groups were performed using Student t test. P < 0.05 was con- time treatments (within 24 hours) with triptolide did not sidered statistically signiﬁcant. reduce P-gp or MDR1 mRNA in KB/VCR cells (Supplementary Fig. S1A–S1C). In contrast, persistent treatments (36 hours or longer) Results reducedP-gpandMDR1 mRNA in KB/VCR and MES-SA/DX5 Triptolide kills MDR cells effectively sublines (Fig. 1D and E), and greater exposure (72 hours) also We evaluated triptolide potency in P-gp–expressing cell reduced P-gp and MDR1 mRNA in K562/A02 cells (Supple- variants, including doxorubicin-selected variants, MES-SA/DX5 mentary Fig. S1D and S1E). Therefore, less P-gp and MDR1 (22), and K562/A02 (2), and vincristine-selected KB/VCR mRNA might be due to transcription inhibition caused by (9). These sublines were resistant to agents used for their degradation of RPB1. After rescuing RPB1 degradation using establishment, respectively as depicted in Fig. 1B. Triptolide a CDK7-selective inhibitor, BS-181, reduced P-gp was rescued potently killed MDR sublines and was 2-fold more potent in in KB/VCR cells (Supplementary Fig. S1F). In addition, short MDR MES-SA/DX5 and KB/VCR sublines but approxi- treatments did not change accumulation of the P-gp substrate mately equipotent in K562 and K562/A02 cell lines and the Rh123 (Fig. 1F). Thus, triptolide is potent and broad spectrum resistance factor is in Fig. 1C. Triptolide reduced RPB1 in time- for overcoming tumor drug resistance, and this activity is

RF Vincristine Adriamycin MES-SA/DX5 AB 36.67 MES/SA K562/A02 K562 163.86 O 0 5 10 15 20 25 30 KB/VCR O KB 40 OH 0 0.5 1 O μ O C IC50 ( mol/L) H RF

O MES-SA/DX5 0.54 Triptolide Figure 1. MES/SA Triptolide kills MDR tumor cells. A, the K562/A02 1.23 chemical structure of triptolide. B, K562 MDR sublines were resistant to drugs that were used for their establishment. KB/VCR 0.55 C, triptolide elicited potent cell killing KB in MDR sublines and respective D 0 10 20 30 40 parental cell lines. Data from three IC50 (nmol/L) independent experiments are expressed as mean SD. The KB/VCR MES-SA/DX5 K562/A02 resistance factor (RF) was calculated Triptolide (50 nmol/L) 0 36 48 60 0 36 48 60 0364860(h) as the ratio of IC50 value of MDR cells to that of corresponding parental cells. RPB1 D and E, three MDR cell lines were treated with triptolide at 50 nmol/L for P-gp indicated time. Cells were lysed and immunoblotted for RPB1 and P-gp (D), GAPDH or RNA was extracted and MDR1 mRNA was measured (E). F, triptolide did not affect the efﬂux of Rh123. Cells E KB/VCR F KB KB/VCR were treated as indicated in Materials MES-SA/DX5 1 Control and Methods and assessed with ﬂow 5 cytometry. K562/A02 2 Rh123 6 1.4 3 Rh123 + Verapamil 7 MDR1 1.2 4 Rh123 + Triptolide 8

1.0 80 3 6 2 0.8 0.6 1 8

40 7 0.4 Counts 5 4 0.2

0.0 0 0 1 2 Relative mRNA level of Relative mRNA 0 36 48 60 (h) 10 10 10 103 Triptolide (50 nmol/L) Fluorescence

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independent of drug transporters, such as P-gp. However, motherapeutics (24, 25). Cancer stem cells within some speciﬁc differences in reducing P-gp expression in different MDR sub- tumors may be able to self-renew (26), and colony formation lines might explain different sensitivities to triptolide, possibly assays could be used to reﬂect this. We observed that MDR by removing the inhibition of P-gp on caspases (23). KB/VCR cells had enhanced colony formation (10%) compared with parental KB cells. However, triptolide inhibited colony Triptolide inhibits colony formation and downregulates formation of KB/VCR more potently than that of KB cells, (IC50, c-MYC in KB and MDR KB/VCR cells 0.68 and 0.41 nmol/L respectively; Fig. 2A and Supplementary In addition to overexpression of drug transporters, cancer stem Fig. S2A). Therefore, MDR KB/VCR cells are more sensitive to cells have been proposed to contribute to tumor MDR to che- triptolide than parental KB cells, similar to that shown in Fig. 1C.

Triptolide (nmol/L, 72 h)

Triptolide (50 nmol/L) Figure 2. c-MYC Triptolide differentially inhibits colony formation of MDR KB/VCR and parental KB cells and the expression of c-MYC, OCT-4, SOX-2, and NANOG. A, KB and KB/VCR cells were cultured as indicated in Materials and Methods, and colonies were ﬁxed, stained, and photographed with ImageQuant LAS 4000. B, KB and KB/VCR cells were as treated as indicated and immunoblotted for c-MYC, OCT-4, SOX-2, NANOG, and GAPDH. C and D, comparisons of c-MYC protein (C) and c-MYC mRNA (D) in MDR sublines and respective parental cell lines. E, KB and KB/VCR cells were transfected as indicated in Materials and Methods and immunoblotted for c-MYC and

c-MYC mRNA GAPDH. F, KB and KB/VCR cells were

c-MYC Relative levels of transfected as indicated in Materials and Methods and 72-hour IC50s were measured using SRB assay. Data from three independent experiments are expressed as mean SD. Triptolide (50 nmol/L, h)

pMSCV-c-MYC pMSCV-c-MYC c-MYC

c-MYC

Triptolide IC50 (nmol/L)

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Transcription factors such as c-MYC, SOX-2, OCT-4, and residueisexactlytheserineresidueatthe5thsiteofthe NANOG are critical for cancer stem cells (27). Both KB and standard CTD repeat located between the 1874th and 1880th KB/VCR cells highly express c-MYC, SOX-2, and OCT-4 proteins amino acid residues of the RPB1 protein (13). Therefore, here, but express low NANOG protein (Fig. 2B). Triptolide reduced we defined a precise Ser5 site in CTD repeats, that is, Ser1878, c-MYC in a time-dependent manner but only slightly lowered which can be phosphorylated by triptolide-activated CDK7. SOX-2 and did not change OCT-4 or NANOG in these cells Rickert and colleagues reported that CDK7 could phosphor- (Fig. 2B). c-MYC mRNA consistently decreased in triptolide- ylate the Serine 5 at the consensus heptapeptide YSPTSPX treated cells (Supplementary Fig. S2B). Similarly, triptolide in vitro by synthesizing different types of peptides, including reduced c-MYC in other cells (Supplementary Fig. S2C and YSPTSPT (30). As the sequence coverage in the mass spectrom- S2D). Notably, all MDR cells expressed more c-MYC protein etry analysis is only 33% (Supplementary Fig. S3) and lack and c-MYC mRNA than their corresponding parental cells of lysines in the CTD resulted in poor trypsin digestions, we (Fig. 2C and D and Supplementary Fig. S2B). Thus, tripto- failed to identify other phosphorylated Ser5 site in the CTD lide-driven reduction of c-MYC expression might contribute to repeats. cell killing or enhanced sensitivity of MDR KB/VCR cells. However, ectopic expression of c-MYC did not confirm this XPB does not contribute to RPB1 degradation or cell killing because increased c-MYC did not significantly reverse cell induced by triptolide killing caused by triptolide in KB and KB/VCR cells (Fig. 2E Triptolide can bind to XPB at its Cys342 residue due to a and F). Therefore, exogenous c-MYC might not rescue the cell covalent modification by the 12,13-epoxide group of triptolide killing of triptolide, either in MDR cells or parental cells. (15, 16). However, a Cys342 mutation of XPB to threonine, but not to alanine or to serine, could rescue cell killing of triptolide Triptolide activates CDK7 by phosphorylating Thr170, which (16, 17), challenging the notion that XPB contributes to trip- leads to phosphorylation of RPB1 at Ser1878 tolide-driven RPB1 degradation and cell killing. Smurnyy and Cell-killing activity of triptolide is correlated with CDK7- colleagues studied the relationship between XPB mutations and mediated RPB1 degradation (13), and this may be true for cell killing induced by triptolide using CRISPR/Cas9 gene drug-resistant tumor cells as well, because triptolide induced editing technology (31); however, various XPB mutations dif- degradation of RPB1 in drug-resistant MES-SA/DX5 and ferent from the Cys342 residue could lead to triptolide resis- KB/VCR cells in a manner similar to respective parental tance. Silencing XPB with specific siRNA duplexes (siXPB) did MES/SA and KB cells (Fig. 3A). Moreover, pretreatments with not reverse the effects of triptolide on Rpb1 (Fig. 4A) or cell BS-181 (28) reversed triptolide-induced RPB1 degradation proliferation (Fig. 4B) in SK-OV-3 cells. Consistently, triptolide similarlyinbothMDRKB/VCRandparentalKBcellsandin caused similar RPB1 degradation (Fig. 4C) and proliferation ovarian cancer SK-OV-3 cells (Fig. 3B). inhibition (Fig. 4D) in both human XPB-deficient (GM02252) Phosphorylation of CDK7 at Thr170 has been revealed to and XPB-proficient (IM-9) lymphocyte cells. Triptolide also augment phosphorylation of RPB1 at its CTD (29). We induced degradation of RPB1 in GM21071, a human fibroblast observed that triptolide increased phosphorylation of CDK7 cell line with XPB deficiency (Fig. 4E). Thus, XPB does not at Thr170 in SK-OV-3 cells (Fig. 3C), as well as in KB and KB/ contribute to RPB1 degradation or cell killing induced by VCR cells (Fig. 3D), which may contribute to activation driven triptolide, although it covalently binds to XPB. by triptolide. BS-181 rescued the degradation of RPB1 induced by triptolide, so we studied whether BS-181 could rescue cell The p44 subunit of TFIIH is not correlated with RPB1 killing induced by triptolide. Data showed that 10 mmol/L BS- degradation and proliferative inhibition induced by triptolide 181 could partially rescue cell killing after 72 hours of treat- p44, another subunit of TFIIH, possesses E3 ubiquitin ligase ment with triptolide (Fig. 3E) in K562, K562/A02, KB, KB/VCR, activity in yeast (32). Possibly, triptolide causes degradation of and SK-OV-3 cells. RPB1 measurements after combining 10 RPB1 via p44-mediated ubiquitination. To confirm this, the mmol/L BS-181 with 30 nmol/L triptolide for 72 hours in both TALEN technique (Supplementary Fig. S6) was used to eliminate KB and KB/VCR cells confirmed that BS-181 could partially the expression of p44 in SK-OV-3 cells, and different p44-deficient rescue RPB1 degradation (Fig. 3F). Triptolide elicited antitumor clonal cells were generated (no. 26-4, 26-5, 26-10, 26-12, and and anti-MDR activity through a universal mechanism. 26-23; Fig. 5A). However, p44 protein elimination could not Our previous work indicated that triptolide could phosphor- prevent RPB1 degradation induced by triptolide (Fig. 5B). At both ylate Ser5 residue(s) in the CTD repeats of RPB1; however, low (10 nmol/L) and high (100 nmol/L) concentrations, tripto- which Ser5 residue(s) can be phosphorylated by triptolide- lide also caused similar proliferative inhibition in parental activated CDK7 is unclear. Thus, using immunoprecipitation SK-OV-3 cells and the p44-deficient 26-10 or 26-12 clonal cells to enrich RPB1 in control and triptolide-treated SK-OV-3 cells, (Fig. 5C). Although p44-deficient 26-5 clonal cells had enhanced we analyzed the phosphorylated site(s) of RPB1 with high- survival at 72 hours with 10 nmol/L triptolide, similar enhance- resolution mass spectrometry. RPB1 protein sequence (Sup- ments did not occur under other conditions (Fig. 5C). In response plementary Fig. S3) coverage was obtained from control to triptolide treatment, RPB1 was ubiquitinated (and phos- and triptolide-treated SK-OV-3 cells, and changes in molec- phorylated at Ser5) in p44-deficient 26-5 clonal cells, just as in ular mass revealed a mass shift of þ80.65 Da at Ser1878 p44-proficient SK-OV-3 cells (Fig. 5D). These data indicate that within the peptide spanning the residues from 1874 to 1887 p44 does not mediate ubiquitinated degradation of RPB1 caused (1874YSPTSPTYSPTTPK1887) in triptolide-treated cells (Sup- by triptolide. plementary Fig. S4), but not in untreated cells (Supplementary In addition, we show that WWP2, a reported E3 ubiquitin Fig. S5). This mass shift corresponded to the molecular mass of ligase that can mediate ubiquitinated degradation of RPB1 a phosphate group (Fig. 3G). Moreover, the 1878th serine (33), was not responsible for RPB1 degradation in triptolide-

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A Triptolide (1 μmol/L, min ) 0 10203040506090120240 0 10 20 30 40 50 60 90 120 240

MES/SA

MES-SA/Dx5

KB/VCR

RPB1 GAPDH B C D BS-181(10 μmol/L) − − ++ Triptolide (200 nmol/L) Triptolide (200 nmol/L) Triptolide (200 nmol/L, 2 h) − + − + SK-OV-3 KB KB/VCR KB 0 15 3045 60 120 (min) 0 30 60 0 30 60 (min) KB/VCR p-S5-RPB1 p-S5-RPB1 RPB1 SK-OV-3 p-T170-CDK7 p-T170-CDK7

KB CDK7 CDK7

KB/VCR GAPDH Tubulin β -Actin SK-OV-3 E 150 Triptolide Triptolide + BS-181 F KB KB/VCR BS-181(10 μmol/L) − − ++ − − ++ 100 Triptolide (30 nmol/L, 72 h) − + − + − + − + RPB1 (nmol/L) 50

IC 50 Tubulin

KB K562 KB/VCR K562/A02 SK-OV-3 Cell lines G

Heptapeptide consensus sequence in CTD 1.7e+003

y11 y10 y1y2y3y4y5y6y7y8y9 2+ Y S P T S P T Y S P T T P K

y2+ 244.16 b2 b3 b11

1878 y5+543.31 y10-Phospho[S]++ 530.77 Relative intensity (%) y9+ 991.50 b3+ 348.10 y4++ 224.10 y7+ 793.42 y1+ 147.11 y10++ 580.10 y10-Phospho[S]+ 1060.56 y8+ 894.49 y9-Phospho[S]++ 447.10 y9++ 496.26 b11++ 631.35 y6+ 630.1 y11++ 630.35 631.35 y6+ 630.1 y11++ b11++ y3++ 173.46 b2+ 251.10 0 10 20 30 40 50 60 70 80 90 100 100 200 300 400 500 600 700 800 900 1,000 1,100 m/z

Figure 3. Triptolide induces RPB1 degradation in both parental and MDR cell lines and RPB1 phosphorylation at Ser1878. A, MES/SA, MES-SA/MX5, KB, and KB/VCR cells were treated as shown in Materials and Methods and immunoblotted for RPB1. B, SK-OV-3, KB, and KB/VCR cells were pretreated with a CDK7- speciﬁc inhibitor and then assayed for RPB1. C, SK-OV-3 cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. D, KB and KB/VCR cells were treated as indicated and immunoblotted for phosphorylated RPB1 at Ser5 and phosphorylated CDK7 at Thr170. E, cells were cultured as depicted in Materials and Methods, and IC50 values were measured using a Cell Counting Kit-8 (CCK-8) assay. F, KB and KB/VCR cells were cultured as indicated and immunoblotted for RPB1. G, SK-OV-3 cells were treated as indicated and immunoprecipitated RPB1 and separated with SDS-PAGE. Gel bands were excised, digested, and assessed using LC/MS-MS to identify the phosphorylation site of RPB1. m/z, mass-to-charge ratio.

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B 100 A 80 siNC siXPB Triptolide (45 min) 0 100 200 500 1,000 0 100 200 500 1,000 (nmol/L) 60

RPB1 40 IC50 (nmol/L) NC 15.3 XPB 20 siRNA XPB 16.0 Inhibition rate (%) GAPDH 0 1 10 100 1,000 C GM02252 IM-9 Concentration of triptolide (nmol/L) Triptolide (1 μmol/L) 00.51 2 34 00.51 2 3 4(h) RPB1 D 100 XPB 80 GAPDH 60 IC50 (nmol/L) E GM21071 40 IM-9 13.9 Triptolide (1 μmol/L, h) 0 0.5 1 2 3 4 SK-OV-3 GM02252 13.9 20 RPB1 rate (%)Inhibition XPB 0 1 10 100 1,000 β-Actin Concentration of triptolide (nmol/L)

Figure 4. XPB does not contribute to triptolide-driven RPB1 degradation and cell killing. A and B, XPB was silenced with specific siRNA in SK-OV-3 cells, which were treated with triptolide at the indicated concentrations for 45 minutes and then immunoblotted for XPB and RPB1 (A). Other cells were treated with triptolide at the indicated concentrations for 72 hours and then assessed by SRB. The inhibition rate was calculated from three independent experiments (B). C, IM-9 and GM02252 cells were treated with 1 mmol/Ltriptolidefortheindicatedtime,andcellswereimmunoblottedforXPBandRPB1. D, IM-9 and GM02252 cells were treated with triptolide at the indicated concentrations for 72 hours and then assessed by SRB. The inhibition rate was calculated from three independent experiments. E, GM21071 cells were treated with 1 mmol/L triptolide for the indicated time, and cells were immunoblotted for XPB and RPB1. SK-OV-3 cells were used as positive controls. treated WWP2-deficient F9 cells (Supplementary Fig. S7A). reduction of P-gp in K562/A02 cells led to the distinct sensi- Using BRCA1-reexpressed UWB1.289 cells, we confirmed our tivities of triptolide compared with KB/VCR and MES-SA/ previous conclusion that triptolide-induced RPB1 degradation DX5. We also identified that the CDK7-selective inhibitor, was independent of BRCA1 (Supplementary Fig. S7B; ref. 13). BS-181, could partially rescue degradation of RPB1 in KB/VCR The E3 ubiquitin ligase(s) correlated with ubiquitinated deg- cells, which would in turn rescue reduction of P-gp. In addition, radation of RPB1 responding to triptolide, which remains to triptolide did not significantly change SOX-2, OCT-4, and be clarified. NANOG, but it did reduce c-MYC. This did not appear to contribute to its ability to reverse drug resistance. In contrast, Discussion triptolide led to RPB1 degradation in all three tested MDR sublines. Thus, considering our previous results (13), it is We report that triptolide had potent direct cell killing on three reasonable to conclude that triptolide kills MDR tumor cells MDR cell variants (averaged resistance factor of 0.77). P-gp– by driving RPB1 degradation just as it kills their corresponding expressing sublines were derived from the corresponding non- parental cells. P-gp–expressing parental tumor cell lines by selection with dif- Previous work from our laboratory and others' suggested a ferent anticancer drugs (2, 9, 22). We also observed that triptolide possible coordination between XPB, CDK7, and p44, the three nearly equipotently killed human pancreatic cancer Capan-1 and subunits of TFIIH in triptolide-mediated RPB1 degradation Ewing sarcoma SK-ES-1 cells and PARP inhibitor simmiparib- (13, 15–17). We report that triptolide activated CDK7 by selected resistant variants Capan-1/SP (RF: 0.84) and SK-ES-1/SP stimulating its phosphorylation at Thr170 and then led to the (RF: 1.36), and these variants did not express detectable drug phosphorylation of RPB1 at Ser1878, which is located within transporters, including P-gp, MRP1, or BRCP (data not shown). the CTD repeats. However, that either XPB or p44 did not Thus, triptolide offers broad-spectrum resolution of drug resis- seem to contribute to triptolide-induced RPB1 degradation tance in tumor cells, regardless of their tissue source, drug selec- and cell killing was intriguing. Other known E3 ubiquitin tion, or drug transporter status. ligases, such as BRCA1, VHL (13), and WWP2, were not cor- Triptolide had unique effects on MDR1 gene expression in three related with RPB1 degradation induced by triptolide either. MDR cell variants: with persistent treatments (36 hours) Notably, data to confirm whether XPB mediates RPB1 degra- reduced P-gp and MDR1 mRNA in both KB/VCR and MES-SA/ dation and proliferative inhibition induced by triptolide are DX5 sublines, but longer exposure (72 hours) reduced P-gp and inconsistent. Titov and colleagues (15), He and colleagues MDR1 mRNA in K562/A02 cells for unclear reasons. (16), and Titov (17) revealed that triptolide covalently bound Data show that KB/VCR and MES-SA/DX5 were more sensi- to XPB at its Cys342 and thereby inhibited ATPase activity tive to triptolide compared with K562/A02. Tainton and col- and led to proliferative inhibition. The triptolide analogue leagues reported that P-gp could suppress the activation of lacking the C12,13-epoxide or mutation of Cys342 of XPB to caspases and reduce apoptosis caused by many chemothera- threonine dramatically reduced triptolide-induced inhibition peutic drugs (23). Therefore, we speculated more but slower of cell proliferation and ATPase activity of XPB (16). However,

www.aacrjournals.org Mol Cancer Ther; 15(7) July 2016 1501

Yi et al.

AB SK-OV-3 26-5 26-10 26-12 SK-OV-326-4 26-5 26-1026-1226-23 Triptolide (2 h, μmol/L) 0 0.1 1 0 0.1 1 0 0.1 1 0 0.1 1 p44 RPB1 Figure 5. β-Actin GAPDH p44 does not mediate RPB1 degradation and proliferative inhibition induced by triptolide. A, CD expression of p44 protein was 100 SK-OV-3 26-5 eliminated by TALEN in SK-OV-3 cells Triptolide _ _ and five p44-deficient cell clones + + 80 (1 μmol/L, 25 min) were obtained. SK-OV-3 and p44- 60 IP + + + + deficient cells were collected, lysed, and immunoblotted for p44. B, SK- 40 OV-3 cells and p44-deficient 26-5, 250 kD 26-10, and 26-12 clonal cells were 20 treated with triptolide at the

Cell viability Cell viability (%) Ubiquitin indicated concentrations for 2 hours 0 0 24 48 72 and immunoblotted for RPB1. C, cells were treated with triptolide at Triptolide (10 nmol/L, h) 10 or 100 nmol/L for the indicated SK-OV-3 time, and then the cell viability was assessed using SRB assay and 100 26-10 calculated from three independent 26-5 p-S5-RPB1 experiments. D, SK-OV-3 and p44- 80 26-12 250 kD deﬁcient cells were treated with 60 1 mmol/L triptolide for 25 minutes and then immunoprecipitated (IP). 40 Western blot analysis was used to 20 measure ubiquitination and phosphorylation of RPB1. Cell viability Cell viability (%) 0 0 24 48 72 RPB1 Triptolide (100 nmol/L, h) β-Actin

the studies by the authors cited above (15–17) also found that spongoate, (7) and tanshinone I (2, 8), can induce nearly mutation of Cys342 of XPB to alanine or serine did not rescue equipotent tumor cell killing in MDR sublines and their respec- triptolide-induced proliferative inhibition, although these tive parental cell lines. A common feature of these agents is that mutants were resistant to binding and ATPase inhibition by they do not inhibit P-gp drug-efflux function. Another similar- triptolide (17). They did not study the effects of triptolide ity is that their regulation effects on transcription factors, binding to XPB and RPB1 degradation (15–17). Smurnyy and including RPB1, c-Jun, HIF1a, and Stat3, are correlated with colleagues reported that different mutations of XPB could result the killing of MDR tumor cells. However, among these com- in drug resistance to triptolide, but these mutations were all pounds, triptolide is the most potent for overcoming MDR, different from the Cys342 residue that the above-mentioned possibly because RPB1 as a general transcription factor is far authors reported (15–17). Smurnyy and colleagues also did not more critical in regulating transcription of precursor mRNA examine the effects of triptolide binding to XPB and RPB1 than all the other transcription factors. Also, possibly, triptolide degradation. In addition, overexpression of mutants of XPB in apparently reduces P-gp in triptolide-sensitive MDR cells (i.e., WT HCT-116 cells did not induce drug resistance to triptolide MES-SA/DX5 and KB/VCR cells). Because triptolide and its (31). We found that both XPB knockdown by siRNA and XPB analogues are undergoing clinical testing, overcoming MDR deficiency (GM02252 and GM21071, 2 cell lines with different may assist with its clinical development. Our data offer a tissue origins, commercially available) did not ease RPB1 molecular mechanism of triptolide-driven RPB1 degradation, degradation and proliferative inhibition induced by triptolide. which may inform future studies to offer new molecular target Thus, binding of triptolide to XPB is not a critical factor for (s) for overcoming MDR. inducing RPB1 degradation and subsequent cell killing. In contrast, all current evidence reveals that triptolide drives Disclosure of Potential Conflicts of Interest RPB1 degradation via CDK7-mediated Ser5 phosphorylation No potential conflicts of interest were disclosed. and subsequent ubiquitination, which is responsible for the proliferative inhibition induced by triptolide. Therefore, find- Authors' Contributions ing kinase(s) and E3 ubiquitin ligase(s) that are respectively Conception and design: J.-M. Yi, Y.-Q. Wang, Z.-H. Miao responsible for triptolide-driven CDK7 activation via phos- Development of methodology: J.-M. Yi phorylation at Thr170 and RPB1 ubiquitination will be the Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Zhou, Z.-H. Miao focus of future investigations. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, In addition to triptolide, many other natural products, in- computational analysis): J.-M. Yi, Z.-H. Miao cluding salvicine (3, 4), pseudolaric acid B (5, 6), methyl Writing, review, and/or revision of the manuscript: J.-M. Yi, Y.-Q. Wang, Z.-H. Miao

1502 Mol Cancer Ther; 15(7) July 2016 Molecular Cancer Therapeutics

Triptolide Kills MDR Tumor Cells

Administrative, technical, or material support (i.e., reporting or organizing Foundation of China (no. 81321092; to Z.H. Miao), and the State Key Labo- data, constructing databases): X.-J. Huan, S.-S. Song, Z.-H. Miao ratory of Drug Research. Study supervision: Y.-Q. Wang, Z.-H. Miao The costs of publication of this article were defrayed in part by the pay- ment of page charges. This article must therefore be hereby marked advertise- ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Grant Support This work was supported by the National Basic Research Program of Received September 11, 2015; revised March 16, 2016; accepted March 19, China (no. 2012CB932502; to Z.H. Miao), the National Natural Science 2016; published OnlineFirst March 29, 2016.

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www.aacrjournals.org Mol Cancer Ther; 15(7) July 2016 1503

Triptolide Induces Cell Killing in Multidrug-Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44

Jun-Mei Yi, Xia-Juan Huan, Shan-Shan Song, et al.

Mol Cancer Ther 2016;15:1495-1503. Published OnlineFirst March 29, 2016.

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