The covalent CDK7 inhibitor THZ1 enhances temsirolimus-induced cytotoxicity via autophagy suppression in human renal cell carcinoma

Cancer Letters 471 (2020) 27–37

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Original Articles

The covalent CDK7 inhibitor THZ1 enhances temsirolimus-induced cytotoxicity via autophagy suppression in human renal cell carcinoma

T

Po-Ming Chowa,b,c,1, Shing-Hwa Liua,d,e,1, Yu-Wei Changb,c, Kuan-Lin Kuoa,b,c, Wei-Chou Linf, Kuo-How Huangb,c,∗ a

Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipe, 100, Taiwan Department of Urology, National Taiwan University Hospital, Taipei, 100, Taiwan c Department of Urology, College of Medicine, National Taiwan University, Taipe, 100, Taiwan d Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, 404, Taiwan e Department of Pediatrics, National Taiwan University Hospital, Taipe, 100, Taiwan f Department of Pathology, National Taiwan University Hospital, Taipe, 100, Taiwan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Genitourinary cancer Drug resistance Apoptosis Cell cycle mTOR inhibitor

Renal cell carcinoma (RCC) is a major cancer of the kidney. The 5-year survival rate is overall 74% and only 8% for Stage 4 cancers. Several agents including tyrosine kinase inhibitors, mTOR inhibitors, and immune checkpoint inhibitors are available as first- or second-line therapy for metastatic RCC. However, the survival benefits are limited. Recently, THZ1 has been identified as a cyclin-dependent kinase 7 (CDK7) inhibitor that interferes with the transcriptional machinery. Although it is apparently effective in various cancer models, the data for RCC has never been reported. In this study, we demonstrated the impact of CDK7 expression on tumor progression and patient survival in a clinical cohort. We found that THZ1 induced apoptosis and cell cycle arrest in RCC cells, thereby reducing cell viability. Furthermore, THZ1 acted synergistically with temsirolimus in vitro, probably by inhibiting autophagy. Moreover, compared to either THZ1 or temsirolimus used individually, the combination treatment further suppressed tumor growth in vivo. These results indicate that CDK7 is associated with the progression and prognosis of RCC, and is a potential therapeutic target for overcoming drug resistance in this cancer.

1. Introduction

endothelial growth factor (VEGF) [2]. In the absence of the genetic hallmarks of solid tumors—such as TP53 or KRAS mutations [3]—angiogenesis is the main target for clear cell RCC systemic therapy. Treatment options for metastatic RCC (mRCC) are quite limited. Chemotherapy, radiotherapy, and hormonal therapy have little effect on mRCC. INF-α and high-dose IL-2 were the only systemic therapies available before the era of targeted agents, and they were associated with low response rates and profound toxicity [4,5]. During the past 10 years, anti-angiogenic agents targeting VEGF and mTOR have become the mainstream treatments [6–11], and an immune checkpoint inhibitor was approved in 2015 [12]. Although there are several potential options for both first- and second-line therapies based on phase 3 trials, the objective response rate is generally less than 30%, providing 3 to 5-

Renal cell carcinoma (RCC) is the major cancer of the kidney. According to the Surveillance, Epidemiology, and End Results (SEER) Program database, the 5-year overall survival (OS) rate is 74% for all stages of RCC. Stage 1 and Stage 2 cancers—which account for more than 2/3 of cases—typically have a good OS rate of over 90%, whereas the OS rates for stage 3 and 4 cancers are 53% and 8%, respectively. Clear cell RCC is the most common subtype (70%) of RCC. The risk factors for RCC include: mutations of the von Hippel–Lindau tumor suppressor gene (VHL), protein polybromo-1 gene (PBRM-1) [1]; and dysregulation of the hypoxia-inducible factor (HIF) and the downstream hypoxia-driven genes, including the genes that encode vascular

Abbreviations: CDK7, cyclin-dependent kinase 7; CTD, carboxyl-terminal domain; ICI, immune checkpoint inhibitor; mRCC, metastatic RCC; RCC, renal cell carcinoma ∗ Corresponding author. Department of Urology, National Taiwan University Hospital and College of Medicine, National Taiwan University, No. 7, Zhongshan S. Rd., Zhongzheng Dist, Taipei, 100, Taiwan. E-mail addresses: [email protected] (P.-M. Chow), [email protected] (S.-H. Liu), [email protected] (Y.-W. Chang), [email protected] (K.-L. Kuo), [email protected] (W.-C. Lin), [email protected] (K.-H. Huang). 1 These authors made equal contributions to this work and share first authorship. https://doi.org/10.1016/j.canlet.2019.12.005 Received 9 August 2019; Received in revised form 18 November 2019; Accepted 3 December 2019 0304-3835/ © 2019 Elsevier B.V. All rights reserved.

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month survival benefits over a placebo [6–11]. The mTOR pathway is involved in tumor proliferation via increasing protein synthesis [13]. It is also involved in angiogenesis via the activation of HIF-1α and VEGF [14]. Two mTOR inhibitors (temsirolimus and everolimus) have been approved for first- and second-line treatment of mRCC. However, their clinical effects are unsatisfactory, and drug resistance is common. Notably, temsirolimus remains the only first-line targeted therapy specifically for poor-risk RCC patients [7]. In addition to the discovery of new targets for therapy, combining agents with different mechanisms is also a common strategy for overcoming drug resistance. Kwiatkowski et al. carried out cell-based screening and identified THZ1, a selective covalent inhibitor of cyclin-dependent kinase 7 (CDK7). THZ1 targets a secluded cysteine residue outside the canonical kinase domain, and is therefore selective for CDK7 [15]. CDK7 phosphorylates serine-5 (Ser5) on the carboxyl-terminal domain (CTD) of RNA Pol II, and induces subsequent reactions including gene transcription, mRNA capping and methylation, and promoter proximal pausing [16]. The covalent binding of THZ1 to CDK7 results in the dysfunction of CDK7 during the phosphorylation of RNA Pol II, without altering the expression of CDK7. These investigations indicate that the specific and selective inhibition of CDK7 may reduce the side effects associated with the treatment of clinical tumors by exerting potent cytotoxic activity against tumor cells but not normal cells. THZ1 was shown to be highly effective in killing tumors without targetable driver mutations—including neuroblastomas, small cell lung cancer, and triple-negative breast cancer—resulting in a substantial reduction in tumor volume by inducing apoptosis and suppressing cell proliferation [15,17,18]. Although THZ1 has been studied in various tumors, its antitumor effect has not been reported in RCC. Combinations of THZ1 and other targeted agents have also not been reported. Because patients with mRCC usually have a short life expectancy of approximately 2 years, most patients are not afforded the opportunity of receiving more than two lines of treatment before death. The chance of drug resistance may be reduced by combining drugs with different mechanisms. In the present study, we investigated the therapeutic effect of THZ1 in RCC, both when used individually or in combination with temsirolimus, as well as the underlying mechanism involved.

board (IRB) approved the present study and waived the informed consent requirement. The IRB case number is 201802014RIND.

2. Materials and methods

We obtained 786-O (#60243) and A-498 (#60241) cell lines from the Bioresource Collection and Research Center (BCRC), Taiwan. Caki-2 (#HTB-47) and ACHN (#CRL-1611) cell lines were obtained from the American Type Culture Collection (ATCC), USA. The 786-O and Caki2 cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) (#11995-040, Gibco®, Thermo Fisher Scientific Inc., Waltham, MA, USA). The ACHN and A-498 cells were cultured in Minimum Essential Medium (MEM) (#11095-072, Gibco®). Both media were supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml) (#15140-122, Gibco®), and Lglutamine (2 mM) (#A2916801, Gibco®). All cells were cultured at 37 °C in a humidified 5% CO2 atmosphere; the cells were subcultivated with 0.05% trypsin–ethylenediaminetetraacetic acid (EDTA) (#15400054, Gibco®) when they reached ~90% confluence.

2.3. Patient data We reviewed patients who had received radical or partial nephrectomy for renal tumors of clinical stage 2 or greater between 2013 and 2018 at our institute. Only patients with clear cell RCCs were included. These patients were followed until May 31, 2019. We collected the age, sex, and survival status data from electronic medical records and the cancer registry database. Overall survival was calculated from the day of diagnosis to the day of death. We subjected specimens from the nephrectomies to immunohistochemistry (IHC) staining to determine the expression levels of CDK7. 2.4. Histology and immunohistochemistry (IHC) The tissue specimens were fixed in 10% formalin and embedded in paraffin for histologic examination. We deparaffinized a series of 4-μm paraffin sections using by EZ prep (Ventana Medical Systems, Inc., Tucson, AZ, USA), and pretreated each section with Cell Conditioning 1 (CC1) solution (Ventana Medical Systems, Inc., Tucson, AZ) for 32 min. The slides were incubated with anti-human CDK7 (#2916, Cell Signaling Technology, Danvers, MA) for 32 min using an automated Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, AZ). We detected labeling with an Optiview DAB Detection Kit (Ventana Medical Systems, Inc.), as per the manufacturer's protocol. All the sections were counterstained with the hematoxylin in the Ventana reagent. A board-certified pathologist (W.C. Lin), who specialized in uro-oncology and was unaware of the clinical data, evaluated the immunoreactivity of CDK7 and determined the IHC score. The staining intensity was graded as: 0 (negative), 1 (weak staining), 2 (moderate staining), or 3 (strong staining). The mean percentage of positive result in the tumor cells was determined by counting at least 10 random fields at both 40 and 400 magnification. The IHC score was calculated by multiplying the intensity grade and the percentage of positive staining. 2.5. Cell culture

2.1. Reagents and antibodies We purchased THZ1 (#M5228) from AbMole BioScience Inc. (Houston, TX, USA). The BrdU cell proliferation assay kit (#2752) was purchased from Merck KGaA (Darmstadt, Germany), and temsirolimus was purchased from Cayman Chemical (Ann Arbor, MI, USA). The PARP (#9542), cleaved PARP (#9541), caspase-3 (#14220), cleavedcaspase-3 (#9664), caspase-7 (#12827), cleaved caspase-7 (#8438), cleaved caspase-8 (#9496), caspase-9 (#9508), cleaved caspase-9 (#9501), β-actin (#4970), cyclin D1 (#2978), cyclin D3 (#2936), CDK4 (#2906), CDK6 (#3136), p27 (#3686), PCNA (#13110), phospho-cdc25C (Ser216) (#4901), cdc25C (#4688), LC3B (#3868), Atg5 (#12994), Atg12 (#4180), phospho-Rpb1 CTD (Ser2) (#13499), phospho-Rpb1 CTD (Ser5) (#13523), phospho-Rpb1 CTD (Ser2/Ser5) (#13546), phospho-Rpb1 CTD (Ser7) (#13780) and Rpb1 NTD (#14958) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). We purchased the caspase-8 (#ALX-804-242C100) antibody from Enzo Life Sciences, Inc. (Farmingdale, NY, USA), and the cdc2 (#1161-1) and p62 (#3340-1) antibodies from Epitomics, Inc. (Burlingame, CA, USA).

2.6. Cell viability assay The cells were seeded at a density of 60% in 6-well plates, and grown overnight. They were then either treated with THZ1 and temsirolimus for the indicated time, or received no treatment. After incubation, the viable cells were identified using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (#21795, Cayman Chemical) assay as follows: 1 ml of the MTT solution (0.5 mg/ ml) was added to each well, which was incubated for 1 h at 37 °C. Next, the supernatant was aspirated, and the MTT-formazan crystals formed by metabolically viable cells were dissolved in 1 ml of dimethyl sulfoxide (DMSO). Finally, the absorbance at 560 nm was monitored using

2.2. Ethics statement The present research meets all applicable standards for the ethics of experimentation and research integrity. The local institutional review 28

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Fig. 1. CDK7 is an unfavorable factor in renal cancer patients (A) Immunohistochemical staining of two clinical tumor samples. The tumor parts and normal parts from the nephrectomy specimen were stained with anti-CDK7 antibody. (B) Immunohistochemical staining of tumor samples from patients of different clinical stages. The tumor samples were stained with the anti-CDK7 antibody. The tissue sections were digitized at 200 × magnification. (C) CDK7 expression score of 83 tumors according to their clinical stages. The data are presented as mean ± SD and were analyzed using two-tailed Student's t-tests. (D) Kaplan–Meier curve for the overall survival of 83 patients according to the level of CDK7 expression in their tumor samples. The data are presented as mean ± SD and were analyzed using a log-rank (Mantel–Cox) test.

previous studies [20]. The combined dose effects were described as the median, dose, and CI effects. CI values less than, equal to, and greater than 1 were taken to indicate synergistic, additive, and antagonistic effects, respectively [19].

a Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific Inc.) [34].

2.7. Combination index 2.8. Immunoblotting

The combined effects of THZ1 and temsirolimus were determined using CalcuSyn software (version 1.1.1, Biosoft, Cambridge, UK), a program based on the method described by Chou and Talalay [19]. The effect at a THZ1 to temsirolimus ratio of 1:200 was evaluated through median-effect and combination index (CI) analysis, as described in

All experiments were performed using standard protocols [34]. Briefly, the cells were lysed in Gold Lysis Buffer containing protease and phosphatase inhibitors (10% glycerol, 1% Triton X-100, 20 mM Tris29

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Fig. 2. THZ1 induces apoptosis in human renal cell carcinoma (RCC) cells (A, B) The 786-O and Caki-2 cells were treated with THZ1 at the indicated doses and times, and cell viability was then determined by a MTT assay. The effect of THZ1 is dose- and time-dependent in RCC cells. (C, D) The 786-O and Caki-2 cells were treated with THZ1 for 24 h, and the cell distribution was analyzed by flow cytometry. THZ1 increased the expression of Annexin V in both types of cells. Each bar represents the mean ± SD (n ≥ 3). The asterisks represent statistically significant differences from the control group (**p < 0.01; ***p < 0.001). (E, F) The cells were treated with THZ1 for 24 h, then the expression of apoptosis-related proteins was determined by immunoblotting. β-actin was used as an internal control.

3. Results

HCl, 137 mM NaCl, 1 mM ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N'-tetraacetic acid (EGTA), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml Leupeptin, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM sodium pyrophosphate, 100 μM β-glycerophosphate, pH 7.9). Quantified cell lysates were mixed with 5X sodium dodecyl sulfate (SDS) sample buffer (10% SDS, 250 mM Tris-HCl, 50% glycerol, 5 mg/mL bromophenol blue, and 50 μl/mL β-mercaptoethanol, pH 6.8, #TAAR-TB2, Biotools, Taipei, Taiwan). The samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life-Sciences, Chicago, IL, USA). The membrane was blocked with 2.5% bovine serum albumin (BSA)/phosphate-buffered saline (PBS) buffer and immunoblotted with various primary antibodies overnight at 4 °C with gentle rotation. We then incubated the membrane with horseradish peroxidase (HRP)-conjugated secondary antibodies (#GTX213110-01, #GTX213111-01, GeneTex) diluted in TBST buffer (a mixture of Tris-buffered saline and Tween 20) for 1 h at 25 °C. Finally, we detected the signals using Immobilon™ Western Chemiluminescent HRP Substrate (ECL) (#WBKLS0500, Merck KGaA), obtained images of the blots using an ImageQuant™ LAS 4000 system, and analyzed the images using ImageQuant™ TL8.1 software (GE Healthcare Life-Sciences).

3.1. CDK7 expression was increased in renal cell carcinoma To establish the significance of CDK7 expression in RCC formation, we subjected the clinical samples to IHC staining. A total of 83 patients who had undergone nephrectomy were included in our clinical cohort. These patients were divided into two groups according to the level of CDK7 expression in their tumors. Age and sex distribution were similar in the CDK7-high and CDK7-low groups, whereas the clinical stages were significantly higher in the CDK7-high group (Table S1). We compared CDK7 expression levels in tumor specimens to those from neighboring normal tissues from the same nephrectomy patients (Fig. 1A). The tumors exhibited higher CDK7 expression than the normal tissues. Tumors of different stages were compared for their CDK7 expression. As shown in the representative photos (Fig. 1B), CDK7 expression increased as the tumor stage increased. When the tumors were divided into the localized (stages I and II) and advanced (stages III and IV) groups, the expression of CDK7 was significantly higher in the advanced stage tumors (Fig. 1C). In our clinical cohort, the overall survival was significantly shorter in patients with higher CDK7 expression in the tumors (Fig. 1D). Non-cancer death occurred in only one patient during the follow-up period, so cancer-specific survival should have been very similar to overall survival. Because CDK7 expression is related to cancer stage—which could be the main risk factor for overall survival—we performed multivariate analysis using the Cox regression model for overall survival. After controlling possible confounders including age, sex, and stage, CDK7 expression remained an independent risk factor for overall survival (Table S2). These results suggest that CDK7 may be related to tumor formation, tumor progression, and patient prognosis.

2.9. Flow cytometry The cell population was determined by flow cytometry as follows. After exposing the cells to THZ1 for the indicated time, we washed them with PBS, then stained them with a Muse® Annexin V and Dead Cell Assay Kit (#MCH100105), a Muse® Caspase-3/7 Assay Kit (#MCH100108), or a Muse® Cell Cycle Assay Kit (#MCH100106) according to the manufacturer's protocols. We quantified the stained cells using a Muse® Cell Analyzer (Luminex Co., Austin, TX, USA) equipped with Muse Analysis software (version 1.6.0.0).

3.2. CDK7 inhibition reduced cell viability and induced apoptosis in human RCC cells

2.10. RCC xenograft model Previous studies have shown that the phosphorylating carboxylterminal domain (CTD) of RNA polymerase II (RNAPII) is suppressed by THZ1. We confirmed that the phosphorylation of RNAPII subunit B1 (Rpb1) CTDs was reduced following THZ1 treatment in both 786-O and Caki-2 cells (Fig. S1). To evaluate the cytotoxic effect of THZ1 in RCC cells, we performed an MTT assay to estimate the viability of 786-O, Caki-2, ACHN, and A-498 cells after treatment with various concentrations of THZ1 for 24 h and 48 h (Fig. 2A and B, S2A, and S2B). Cell viability decreased in a dose-dependent manner. The viability further decreased as the treatment duration increased. For the 786-O cells, the half maximal inhibitory concentration (IC50) was not reached after 24 h and was approximately 150 nM after 48 h. The required dose was higher for 786-O cells than for Caki-2 cells. These results indicate that THZ1 has a cytotoxic effect on RCC cells. To evaluate the effect of THZ1 on apoptosis in RCC cells, we carried out flow cytometry with Annexin V staining in 786-O cells after treatment with THZ1 for 24 h (Fig. 2C). The proportion of apoptotic cells increased from 9.3% to 39.5% as the concentration of THZ1 was increased to 200 nM. Similar results were observed in the Caki-2 cells (Fig. 2D). We also confirmed the pro-apoptotic effect of THZ1 by caspase-3/7 staining after THZ1 treatment (Figs. S3A and S3B). Furthermore, we carried out immunoblotting to evaluate the changes in the

All animal care and experimental procedures were conducted in accordance with the protocols approved by the Institutional Animal Care and Use Committee. We suspended 1 × 106 786-O or Caki-2 cells in 100 μL of serum-free medium and mixed with an equal volume of Matrigel® Matrix (Corning Inc., Oneonta, NY, USA), and injected them subcutaneously into 8-week-old nu/nu mice, which we obtained from the National Laboratory Animal Center (Taipei, Taiwan). After 2 weeks of tumor growth, we randomly assigned the mice to experimental and control groups, and treated them with saline or THZ1 and Torisel® (Pfizer Inc., New York, NY, USA) for 4 weeks. We measured the tumor volumes with calipers twice every week, using the formula: V = (π/ 6) × [(A + B)/2]3, where V is the tumor volume, and A and B are the longest and shortest tumor diameters, respectively. 2.11. Statistical analysis We performed all the statistical analyses using GraphPad Prism 5 software. The data are presented as means ± SD, or means ± SEM, and were analyzed using two-tailed Student's t-tests, log-rank (Mantel–Cox) tests or one-way analysis of variance (ANOVA). p-values of < 0.05 were considered statistically significant. 31

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Fig. 3. THZ1 inhibits the proliferation of RCC cells by inducing G1 cell cycle arrest (A, B) The 786-O and Caki-2 cells were treated with THZ1 for 24 h after 24-h starvation, and the cell distributions were determined by flow cytometry. (C, D) The cells described in (A) and (B) were lysed and immunoblotted with the indicated antibodies. β-actin was used as an internal control. (E) The level of cell proliferation was quantified using a BrdU assay after treating the 786-O and Caki-2 cells with THZ1 for 24 h. Each bar represents the mean ± SD (n ≥ 3). The asterisks represent statistically significant differences from the control group (**p < 0.01; ***p < 0.001).

Fig. 4. The synergistic effect of THZ1 and temsirolimus on human RCC cells. The indicated dosages of THZ1 and temsirolimus were used to treat 786-O (A) and Caki-2 (B) cells for 24 h, then cell viability was determined by a MTT assay. Each bar represents the mean ± SD (n ≥ 3). The asterisks represent statistically significant differences from the control group (#p < 0.01; *p < 0.001). (C) The synergistic effect was confirmed by a cell viability assay (n ≥ 3), and the combination index (CI) data were generated using CalcuSyn software. CI values are presented in the CI–effect plot (as log10CI ± SD) and CI < 1, = 1, and > 1 indicate synergistic, additive, and antagonistic effects, respectively.

(Fig. 3C and D). Consistently, the results revealed dose-dependent reductions in cyclin D1, cyclin D3, CDK4, CDK6, Cdc2, PCNA, and cdc25c, and a dose-dependent increase in p27. To evaluate the result of cell cycle arrest, we carried out a BrdU assay to estimate cell proliferation activity (Fig. 3E). The ability to proliferate decreased in both types of cells after THZ1 treatment. Collectively, these results indicate that THZ1 reduces the growth of RCC cells by regulating the cell cycle.

amount of protein after THZ1 treatment (Fig. 2E and F). In both 786-O and Caki-2 cells, the inactive forms of apoptotic proteins (caspase-3, 7, 8, 9, and PARP) decreased, whereas the active, cleaved forms increased. Moreover, the apoptotic effect increased after 48 h (Figs. S3C–S3F). These results confirm that THZ1 induces apoptosis in RCC cells.

3.3. CDK7 inhibition resulted in cell cycle retardation in human RCC cells 3.4. CDK7 inhibitor enhanced the cytotoxic effect of temsirolimus by inhibiting autophagy

To evaluate the effect of THZ1 on cell cycle regulation, we carried out flow cytometry to estimate the proportion of cells in the various phases of the cell cycle. THZ1 treatment led to an increased proportion of cells in the G0/G1 phase in both the 786-O and Caki-2 cells as the dose was increased (Fig. 3A and B). We observed similar results in the ACHN and A-498 cells (Figs. S4A and S4B). Furthermore, we carried out immunoblotting to evaluate the changes in cell cycle-related proteins

Because resistance to temsirolimus is common in RCC patients, we further evaluated the cytotoxic effect of a combination of THZ1 and temsirolimus by carrying out an MTT assay after combining a fixed dose of THZ1 (200 nM) and various doses of temsirolimus (0–50 μM) 33

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Fig. 5. Combined THZ1 and temsirolimus treatment enhances apoptotic activity, and inhibits temsirolimus-induced autophagy in RCC cells The 786-O (A) and Caki-2 (B) cells were treated with the indicated concentrations of THZ1 and temsirolimus for 24 h, and immunoblotted with the indicated antibodies. β-actin was used as an internal control.

The tumor volumes were reduced with either THZ1 or temsirolimus treatment, and there was almost no increase in tumor size in the combined treatment group. There were similar results for the final tumor weights. The body weights of the mice did not change significantly during the treatment period (Fig. S7), indicating no obvious toxicity to the mice. The results suggest that THZ1 in combination with temsirolimus has a better anti-cancer effect than either drug alone.

(Fig. 4A and B). In both cell lines, THZ1 significantly reduced cell viability compared to the cell viability in the groups treated with the same concentrations of temsirolimus alone. When combined with THZ1, a much lower dosage of temsirolimus was required to achieve the same cytotoxic effect. Furthermore, to confirm the synergistic effect of THZ1 and temsirolimus, we tested the two drugs at various concentrations. The 786-O and Caki-2 cells were treated with THZ1 in combination with temsirolimus at a 1:200 ratio for 24 h. The dose of the combination was then tested with THZ1 ranging from 75 to 325 nM and temsirolimus ranging from 15 to 65 μM (Tables S3 and S4). The combination indices were generally smaller than 1, indicating a positive synergistic effect (Fig. 4C and S5). The results demonstrate the synergistic effect of combining THZ1 and temsirolimus, and that there is a potential role for THZ1 in overcoming drug resistance to temsirolimus. To investigate the mechanism underlying the enhanced cytotoxic effect of the THZ1 and temsirolimus combination, we carried out immunoblotting after treating the cells with DMSO, THZ1 alone, temsirolimus alone, or both THZ1 and temsirolimus. First, we examined the change in apoptotic proteins in the various treatment groups. We found that the amount of cleaved PARP increased markedly in the combination group relative to the amounts when either drug was used alone, indicating enhanced apoptosis (Fig. 5A and B, S6A, and S6B). Second, we determined whether autophagy—the well-described mechanism underlying resistance to temsirolimus [21,22]—is involved in the effects of this drug combination. We found that temsirolimus alone markedly increased the expression levels of autophagy-related proteins including LC3, ATG12, and ATG5. Whereas THZ1 alone did not significantly alter the levels of the autophagy markers, the combination of THZ1 and temsirolimus resulted in much lower levels of such markers (Fig. 5A and B, S6A and S6B). These results suggest that THZ1 suppresses temsirolimus-induced autophagy. Combining THZ1 with temsirolimus can elicit a better cytotoxic effect by enhancing apoptosis and repressing autophagy.

4. Discussion mRCC treatment has evolved substantially in the last few years. With the emergence of immune checkpoint inhibitors (ICIs), many options are available for both first-line and second-line therapies. Despite the excitement resulting from the discovery of novel ICIs, the overall response rate is still below 30% for these agents [12]. More recently, researchers have investigated the possibility of combining different ICIs or combining a targeted agent with an ICI, and have reported better treatment outcomes than those for targeted therapy alone [23–25]. The concept of combining different agents to overcome resistance is clinically reasonable, but the various combinations and the underlying mechanism have rarely been investigated at the molecular level. Our results reveal that THZ1 can reduce cell viability in human RCC cell lines, mainly through the induction of apoptosis and cell cycle arrest. CDK7 plays important roles in both cell cycle regulation and RNA synthesis. CDK-activating kinase (CAK) is a trimer that comprises CDK7, cyclin H, and Mat1. CAK alone is a positive regulator of CDK1, CDK2, CDK4, and CDK6, and therefore mediates cell cycle progression through its activation of CDKs. CAK also forms the kinase subcomplex TFIIH, which phosphorylates and activates RNA polymerase II for the initiation and elongation of RNA synthesis [26]. Because CDK7 provides kinase activity for the CAK trimer, the inhibition of CDK7 by THZ1 can result in cell cycle arrest and transcription suppression. A reduction of cell proliferation can be expected in cancer cells that rely on the overtranscription of survival signals, whereas normal cells with normal transcription activity are less affected [17]. Previous studies have revealed a diversity of mechanisms for THZ1 function in various types of cancers. Cayrol et al. found that THZ1 reduced the expression of anti-apoptotic proteins by inhibiting the STAT3 signaling pathway [27]. Li et al. found that CDK7 expression was elevated in triple-negative breast cancer, and that THZ1 induced apoptosis in these cells [28]. Greenall et al. found that THZ1 led to DNA damage and cell cycle arrest in the G2 phase, and also reduced mitochondrial

3.5. Combination treatment with THZ1 and temsirolimus further suppressed tumor growth in mice xenografts models To evaluate the treatment effect of THZ1 alone and in combination with temsirolimus, we inoculated RCC xenografts into athymic nu/nu mice. After 2 weeks, the mice received scheduled intraperitoneal injections of normal saline, THZ1 alone, temsirolimus alone, or THZ1 and temsirolimus combined (Fig. 6A). After 4 weeks, the mice were sacrificed and the tumor volumes and weights were measured (Fig. 6B-E). 34

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Fig. 6. THZ1 and temsirolimus have a synergistic therapeutic effect in a mouse xenograft renal tumor model (A) The timeline represents the treatment of the xenografts with THZ1 and temsirolimus. (B, C) The tumor volume was monitored at the indicated dates. THZ1 + temsirolimus treatment significantly suppressed the growth of 786-O and Caki-2 xenografts. (D, E) The bars represent the weights of the tumors. Each bar represents the mean ± SEM (n ≥ 6). The asterisks represent statistically significant differences from the control group (*p < 0.05; **p < 0.01; ***p < 0.001).

possibly owing to the inhibition of autophagy. The importance of CDK7 expression in clinical settings has rarely been investigated. Our study demonstrated the correlation between CDK7 and overall survival in a clinical cohort, and the impact of CDK7 inhibition on cancer progression. After controlling the major risk factors, including tumor stage, CDK7 expression remained an independent risk factor for overall survival. In conclusion, CDK7 is associated with the progression and prognosis of RCC; it is a potential therapeutic target, and is key to overcoming drug resistance in RCC.

translation and oxidative respiration in high-grade glioma [29]. Cheng et al. found that THZ1 treatment altered the expression of glutaminase 1 (GLS1) isoforms and suppressed cell proliferation in non-small cell lung cancer [30]. Our results were consistent with those of the previous studies, and the effects were best demonstrated by our xenograft model. Furthermore, our results show that THZ1 can reduce autophagy, which has not been reported for this CDK7 inhibitor before. The inhibition of mTOR induces autophagy [21,31] which supports tumor survival and progression [32], thereby limiting antitumor efficacy [33]. Therefore, autophagy may be an important mechanism by which cancer cells escape therapy-induced cell death in RCC after treatment with temsirolimus. Adding an autophagy inhibitor may improve the response of RCC cancer cells to temsirolimus. Our results suggest that THZ1 is more effective when used with temsirolimus,

Author contributions P.-M.C., S.-H.L., Y.-W.C., K.-L.K. and K.-H.H designed the research. P.-M.C. and Y.-W.C. performed the experiments. Y.-W.C. analyzed the 35

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data. K.-L.K. assisted with some experiments. W.-C.L. evaluated the IHC data. P.-M.C. and Y.-W.C. wrote the paper. All authors discussed the results and approved the manuscript.

renal-cell carcinoma, N. Engl. J. Med. 373 (2015) 1803–1813. [13] M. Salvadori, Antineoplastic effects of mammalian target of rapamycine inhibitors, World J. Transplant. 2 (2012) 74–83. [14] E.M. Posadas, S. Limvorasak, S. Sharma, R.A. Figlin, Targeting angiogenesis in renal cell carcinoma, Expert Opin. Pharmacother. 14 (2013) 2221–2236. [15] N. Kwiatkowski, T. Zhang, P.B. Rahl, B.J. Abraham, J. Reddy, S.B. Ficarro, A. Dastur, A. Amzallag, S. Ramaswamy, B. Tesar, C.E. Jenkins, N.M. Hannett, D. McMillin, T. Sanda, T. Sim, N.D. Kim, T. Look, C.S. Mitsiades, A.P. Weng, J.R. Brown, C.H. Benes, J.A. Marto, R.A. Young, N.S. Gray, Targeting transcription regulation in cancer with a covalent CDK7 inhibitor, Nature 511 (2014) 616–620. [16] V. Posternak, M.D. Cole, Strategically targeting MYC in cancer, F1000Research, 2016, p. 5. [17] C.L. Christensen, N. Kwiatkowski, B.J. Abraham, J. Carretero, F. Al-Shahrour, T. Zhang, E. Chipumuro, G.S. Herter-Sprie, E.A. Akbay, A. Altabef, J. Zhang, T. Shimamura, M. Capelletti, J.B. Reibel, J.D. Cavanaugh, P. Gao, Y. Liu, S.R. Michaelsen, H.S. Poulsen, A.R. Aref, D.A. Barbie, J.E. Bradner, R.E. George, N.S. Gray, R.A. Young, K.K. Wong, Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor, Cancer Cell 26 (2014) 909–922. [18] Y. Wang, T. Zhang, N. Kwiatkowski, B.J. Abraham, T.I. Lee, S. Xie, H. Yuzugullu, T. Von, H. Li, Z. Lin, D.G. Stover, E. Lim, Z.C. Wang, J.D. Iglehart, R.A. Young, N.S. Gray, J.J. Zhao, CDK7-dependent transcriptional addiction in triple-negative breast cancer, Cell 163 (2015) 174–186. [19] T.C. Chou, P. Talalay, Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors, Adv. Enzym. Regul. 22 (1984) 27–55. [20] D.R. Vogus, A. Pusuluri, R. Chen, S. Mitragotri, Schedule dependent synergy of gemcitabine and doxorubicin: improvement of in vitro efficacy and lack of in vitroin vivo correlation, Bioeng. Transl. Med. 3 (2018) 49–57. [21] J.A. Martina, Y. Chen, M. Gucek, R. Puertollano, MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB, Autophagy 8 (2012) 903–914. [22] M. Rusan, K. Li, Y. Li, C.L. Christensen, B.J. Abraham, N. Kwiatkowski, K.A. Buczkowski, B. Bockorny, T. Chen, S. Li, K. Rhee, H. Zhang, W. Chen, H. Terai, T. Tavares, A.L. Leggett, T. Li, Y. Wang, T. Zhang, T.J. Kim, S.H. Hong, N. PoudelNeupane, M. Silkes, T. Mudianto, L. Tan, T. Shimamura, M. Meyerson, A.J. Bass, H. Watanabe, N.S. Gray, R.A. Young, K.K. Wong, P.S. Hammerman, Suppression of adaptive responses to targeted cancer therapy by transcriptional repression, Cancer Discov. 8 (2018) 59–73. [23] R.J. Motzer, K. Penkov, J. Haanen, B. Rini, L. Albiges, M.T. Campbell, B. Venugopal, C. Kollmannsberger, S. Negrier, M. Uemura, J.L. Lee, A. Vasiliev, W.H. Miller Jr., H. Gurney, M. Schmidinger, J. Larkin, M.B. Atkins, J. Bedke, B. Alekseev, J. Wang, M. Mariani, P.B. Robbins, A. Chudnovsky, C. Fowst, S. Hariharan, B. Huang, A. di Pietro, T.K. Choueiri, Avelumab plus axitinib versus sunitinib for advanced renalcell carcinoma, N. Engl. J. Med. 380 (2019) 1103–1115. [24] B.I. Rini, E.R. Plimack, V. Stus, R. Gafanov, R. Hawkins, D. Nosov, F. Pouliot, B. Alekseev, D. Soulieres, B. Melichar, I. Vynnychenko, A. Kryzhanivska, I. Bondarenko, S.J. Azevedo, D. Borchiellini, C. Szczylik, M. Markus, R.S. McDermott, J. Bedke, S. Tartas, Y.H. Chang, S. Tamada, Q. Shou, R.F. Perini, M. Chen, M.B. Atkins, T. Powles, K.- Investigators, Pembrolizumab plus axitinib versus sunitinib for advanced renal-cell carcinoma, N. Engl. J. Med. 380 (2019) 1116–1127. [25] B.I. Rini, T. Powles, M.B. Atkins, B. Escudier, D.F. McDermott, C. Suarez, S. Bracarda, W.M. Stadler, F. Donskov, J.L. Lee, R. Hawkins, A. Ravaud, B. Alekseev, M. Staehler, M. Uemura, U. De Giorgi, B. Mellado, C. Porta, B. Melichar, H. Gurney, J. Bedke, T.K. Choueiri, F. Parnis, T. Khaznadar, A. Thobhani, S. Li, E. Piault-Louis, G. Frantz, M. Huseni, C. Schiff, M.C. Green, R.J. Motzer, I.M.S. Group, Atezolizumab plus bevacizumab versus sunitinib in patients with previously untreated metastatic renal cell carcinoma (IMmotion151): a multicentre, open-label, phase 3, randomised controlled trial, Lancet 393 (2019) 2404–2415. [26] C.C. Ebmeier, B. Erickson, B.L. Allen, M.A. Allen, H. Kim, N. Fong, J.R. Jacobsen, K. Liang, A. Shilatifard, R.D. Dowell, W.M. Old, D.L. Bentley, D.J. Taatjes, Human TFIIH kinase CDK7 regulates transcription-associated chromatin modifications, Cell Rep. 20 (2017) 1173–1186. [27] F. Cayrol, P. Praditsuktavorn, T.M. Fernando, N. Kwiatkowski, R. Marullo, M.N. Calvo-Vidal, J. Phillip, B. Pera, S.N. Yang, K. Takpradit, L. Roman, M. Gaudiano, R. Crescenzo, J. Ruan, G. Inghirami, T. Zhang, G. Cremaschi, N.S. Gray, L. Cerchietti, THZ1 targeting CDK7 suppresses STAT transcriptional activity and sensitizes T-cell lymphomas to BCL2 inhibitors, Nat. Commun. 8 (2017) 14290. [28] B. Li, T. Ni Chonghaile, Y. Fan, S.F. Madden, R. Klinger, A.E. O'Connor, L. Walsh, G. O'Hurley, G. Mallya Udupi, J. Joseph, F. Tarrant, E. Conroy, A. Gaber, S.F. Chin, H.A. Bardwell, E. Provenzano, J. Crown, T. Dubois, S. Linn, K. Jirstrom, C. Caldas, D.P. O'Connor, W.M. Gallagher, Therapeutic rationale to target highly expressed CDK7 conferring poor outcomes in triple-negative breast cancer, Cancer Res. 77 (2017) 3834–3845. [29] S.A. Greenall, Y.C. Lim, C.B. Mitchell, K.S. Ensbey, B.W. Stringer, A.L. Wilding, G.M. O'Neill, K.L. McDonald, D.J. Gough, B.W. Day, T.G. Johns, Cyclin-dependent kinase 7 is a therapeutic target in high-grade glioma, Oncogenesis 6 (2017) e336. [30] Z.J. Cheng, D.L. Miao, Q.Y. Su, X.L. Tang, X.L. Wang, L.B. Deng, H.D. Shi, H.B. Xin, THZ1 suppresses human non-small-cell lung cancer cells in vitro through interference with cancer metabolism, Acta Pharmacol. Sin. 40 (2018) 814–822. [31] Y.H. Zhan, J. Liu, X.J. Qu, K.Z. Hou, K.F. Wang, Y.P. Liu, B. Wu, beta-Elemene induces apoptosis in human renal-cell carcinoma 786-0 cells through inhibition of MAPK/ERK and PI3K/Akt/mTOR signalling pathways, Asian Pac. J. Cancer Prev. APJCP 13 (2012) 2739–2744. [32] R.K. Amaravadi, J. Lippincott-Schwartz, X.M. Yin, W.A. Weiss, N. Takebe,

Declaration of competing interest None. Acknowledgment We would like to acknowledge the service provided by the RCF3 and RCF6 Laboratory of the Department of Medical Research at the National Taiwan University Hospital. English Editing was provided by Elsevier Language Editing Services and the Department of Medical Research at the National Taiwan University Hospital. The present work was supported by the Ministry of Science and Technology (MOST), Taiwan (grant number: 107-2314-B-002-268-MY2), and by the National Taiwan University Hospital, Taiwan (grant numbers: 106-3672, 1074077, and 108-4137). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.canlet.2019.12.005. References [1] S. Nabi, E.R. Kessler, B. Bernard, T.W. Flaig, E.T. Lam, Renal cell carcinoma: a review of biology and pathophysiology, F1000Res, vol. 7, 2018, p. 307. [2] N. Cancer, Genome Atlas Research, Comprehensive molecular characterization of clear cell renal cell carcinoma, Nature 499 (2013) 43–49. [3] E. Jonasch, P.A. Futreal, I.J. Davis, S.T. Bailey, W.Y. Kim, J. Brugarolas, A.J. Giaccia, G. Kurban, A. Pause, J. Frydman, A.J. Zurita, B.I. Rini, P. Sharma, M.B. Atkins, C.L. Walker, W.K. Rathmell, State of the science: an update on renal cell carcinoma, Mol. Cancer Res. : MCR 10 (2012) 859–880. [4] G. Fyfe, R.I. Fisher, S.A. Rosenberg, M. Sznol, D.R. Parkinson, A.C. Louie, Results of treatment of 255 patients with metastatic renal cell carcinoma who received highdose recombinant interleukin-2 therapy, J. Clin. Oncol. : Off. J. Am. Soc. Clin. Oncol. 13 (1995) 688–696. [5] J.A. Klapper, S.G. Downey, F.O. Smith, J.C. Yang, M.S. Hughes, U.S. Kammula, R.M. Sherry, R.E. Royal, S.M. Steinberg, S. Rosenberg, High-dose interleukin-2 for the treatment of metastatic renal cell carcinoma : a retrospective analysis of response and survival in patients treated in the surgery branch at the National Cancer Institute between 1986 and 2006, Cancer 113 (2008) 293–301. [6] B. Escudier, T. Eisen, W.M. Stadler, C. Szczylik, S. Oudard, M. Siebels, S. Negrier, C. Chevreau, E. Solska, A.A. Desai, F. Rolland, T. Demkow, T.E. Hutson, M. Gore, S. Freeman, B. Schwartz, M. Shan, R. Simantov, R.M. Bukowski, T.S. Group, Sorafenib in advanced clear-cell renal-cell carcinoma, N. Engl. J. Med. 356 (2007) 125–134. [7] G. Hudes, M. Carducci, P. Tomczak, J. Dutcher, R. Figlin, A. Kapoor, E. Staroslawska, J. Sosman, D. McDermott, I. Bodrogi, Z. Kovacevic, V. Lesovoy, I.G. Schmidt-Wolf, O. Barbarash, E. Gokmen, T. O'Toole, S. Lustgarten, L. Moore, R.J. Motzer, A.T. Global, Temsirolimus, interferon alfa, or both for advanced renalcell carcinoma, N. Engl. J. Med. 356 (2007) 2271–2281. [8] R.J. Motzer, T.E. Hutson, P. Tomczak, M.D. Michaelson, R.M. Bukowski, O. Rixe, S. Oudard, S. Negrier, C. Szczylik, S.T. Kim, I. Chen, P.W. Bycott, C.M. Baum, R.A. Figlin, Sunitinib versus interferon alfa in metastatic renal-cell carcinoma, N. Engl. J. Med. 356 (2007) 115–124. [9] T.E. Hutson, I.D. Davis, J.P. Machiels, P.L. De Souza, S. Rottey, B.F. Hong, R.J. Epstein, K.L. Baker, L. McCann, T. Crofts, L. Pandite, R.A. Figlin, Efficacy and safety of pazopanib in patients with metastatic renal cell carcinoma, J. Clin. Oncol. : Off. J. Am. Soc. Clin. Oncol. 28 (2010) 475–480. [10] R.J. Motzer, B. Escudier, S. Oudard, T.E. Hutson, C. Porta, S. Bracarda, V. Grunwald, J.A. Thompson, R.A. Figlin, N. Hollaender, A. Kay, A. Ravaud, R.S. Group, Phase 3 trial of everolimus for metastatic renal cell carcinoma : final results and analysis of prognostic factors, Cancer 116 (2010) 4256–4265. [11] B.I. Rini, B. Escudier, P. Tomczak, A. Kaprin, C. Szczylik, T.E. Hutson, M.D. Michaelson, V.A. Gorbunova, M.E. Gore, I.G. Rusakov, S. Negrier, Y.-C. Ou, D. Castellano, H.Y. Lim, H. Uemura, J. Tarazi, D. Cella, C. Chen, B. Rosbrook, S. Kim, R.J. Motzer, Comparative effectiveness of axitinib versus sorafenib in advanced renal cell carcinoma (AXIS): a randomised phase 3 trial, The Lancet 378 (2011) 1931–1939. [12] R.J. Motzer, B. Escudier, D.F. McDermott, S. George, H.J. Hammers, S. Srinivas, S.S. Tykodi, J.A. Sosman, G. Procopio, E.R. Plimack, D. Castellano, T.K. Choueiri, H. Gurney, F. Donskov, P. Bono, J. Wagstaff, T.C. Gauler, T. Ueda, Y. Tomita, F.A. Schutz, C. Kollmannsberger, J. Larkin, A. Ravaud, J.S. Simon, L.A. Xu, I.M. Waxman, P. Sharma, I. CheckMate, Nivolumab versus everolimus in advanced

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Cancer Letters 471 (2020) 27–37

P.-M. Chow, et al.

Assoc. Canc. Res. 17 (2011) 3478–3489. [34] K.-L. Kuo, I.-L. Ho, C.-S. Shi, J.-T. Wu, W.-C. Lin, Y.-C. Tsai, H.-C. Chang, C.-T. Chou, C.-H. Hsu, J.-T. Hsieh, S.-C. Chang, Y.-S. Pu, K.-H. Huang, MLN4924, a novel protein neddylation inhibitor, suppresses proliferation and migration of human urothelial carcinoma: In vitro and in vivo studies, Cancer Lett. 363 (2015) 127–136.

W. Timmer, R.S. DiPaola, M.T. Lotze, E. White, Principles and current strategies for targeting autophagy for cancer treatment, Clin. Cancer Res. : Off. J. Am. Assoc. Canc. Res. 17 (2011) 654–666. [33] X.H. Ma, S. Piao, D. Wang, Q.W. McAfee, K.L. Nathanson, J.J. Lum, L.Z. Li, R.K. Amaravadi, Measurements of tumor cell autophagy predict invasiveness, resistance to chemotherapy, and survival in melanoma, Clin. Cancer Res. : Off. J. Am.

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