Amplification of TLK2 Induces Genomic Instability Via Impairing the G 2−M Checkpoint

Published OnlineFirst August 3, 2016; DOI: 10.1158/1541-7786.MCR-16-0161

DNA Damage and Repair Molecular Cancer Research Ampliﬁcation of TLK2 Induces Genomic Instability

via Impairing the G2–M Checkpoint Jin-Ah Kim1,2,3, Meenakshi Anurag1,2,3, Jamunarani Veeraraghavan1,2,3, Rachel Schiff1,2,3, Kaiyi Li1,2,4, and Xiao-Song Wang1,2,3,5,6,7,8

Abstract

Managing aggressive breast cancers with enhanced chromosomal irradiation or doxorubicin. To our knowledge, this is the first report instability (CIN) is a significant challenge in clinics. Previously, we linking TLK2 function to CIN, in contrast to the function of its described that a cell cycle–associated kinase called Tousled-like paralog TLK1 as a guardian of genome stability. This finding yields kinase 2 (TLK2) is frequently deregulated by genomic amplifications new insight into the deregulated DNA damage pathway and þ þ in aggressive estrogen receptor–positive (ER ) breast cancers. In this increased genomic instability in aggressive ER breast cancers. study, it was discovered that TLK2 amplification and overexpression mechanistically impair Chk1/2-induced DNA damage checkpoint Implications: Targeting TLK2 presents an attractive thera- TLK2 fi signaling, leading to a G2–M checkpoint defect, delayed DNA repair peutic strategy for the -ampli ed breast cancers that process, and increased CIN. In addition, TLK2 overexpression mod- possess enhanced genomic instability and aggressiveness. estly sensitizes breast cancer cells to DNA-damaging agents, such as Mol Cancer Res; 14(10); 920–7. 2016 AACR.

Introduction genome-wide copy number aberrations are often used as a surro- þ gate marker to evaluate the level of CIN in cancer (4). Estrogen receptor–positive (ER ) breast cancers (also known as In the presence of DNA damage during S–G phase, cell cycle is luminal breast cancers) account for a vast majority of all breast 2 arrested at G –M checkpoint to ensure the cells to repair their DNA cancers and can be classified into A and B intrinsic subtypes. In 2 before enter into mitosis. The key regulatory step of mitotic entry contrast to the slow-growing and endocrine-sensitive luminal A þ is the activation of Cdk1 via dephosphorylation of Cdk1 at Thr14 tumors, the luminal B tumors are more aggressive form of ER and Tyr15, which is carried out by the Cdc25 phosphatase (5). breast cancers characterized by higher proliferation index and worse G –M checkpoint signaling in response to DNA damage activates clinical outcome after endocrine therapy. Recent large-scale geno- 2 Chk1 and Chk2, which in turn repress Cdc25 phosphatases, mic profiling studies suggest that the markedly enhanced accumu- resulting in the inactivation of CDK1 and cell-cycle arrest at lation of chromosomal aberrations is characteristic of luminal B G –M checkpoint (6). After DNA repair is completed, the mitotic breast tumors (1). Chromosomal instability (CIN) is the major 2 kinases, such as AURKA and PLK1, have a key role in G –M form of genomic instability in human cancers and is characterized 2 checkpoint recovery. In multiple tumors, the amplification and by an increased rate of numerical and structural alterations in the overexpression of AURKA or PLK1 are known causes of G –M chromosomes. CINs have been linked to disease progression, 2 checkpoint defect and enhanced CIN, which is often related to distant metastasis, and therapeutic resistance in breast cancer (1, increased tumor aggressiveness (7). Thus, targeted agents are 2), which pose a great challenge to clinical management. Because of being actively developed against these mitotic kinases, and the mechanistic connection, it is increasingly accepted that numerical Aurora A kinase inhibitors are now in advanced clinical devel- and structural CINs cannot be considered in isolation (3); thus, opments for treating solid tumors (8, 9). It is therefore critical to discover additional genetic aberrations of cell-cycle kinases independent of AURKA or PLK1 that promote G –M checkpoint 1 2 Lester & Sue Smith Breast Center, Baylor College of Medicine, Hous- defects and CIN in the luminal B breast cancers so as to develop ton,Texas. 2Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston,Texas. 3Department of Medicine, Baylor College of Medicine, new targeted therapies. Houston, Texas. 4Department of Surgery, Baylor College of Medicine, In our previous study, we have identified a cell-cycle kinase Houston,Texas. 5Department of Molecular and Cellular Biology, Baylor 6 called "Tousled-like kinase 2" (TLK2) that is targeted for ampli- College of Medicine, Houston, Texas. University of Pittsburgh Cancer fi þ Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. 7Depart- cation in approximately 10.5% of ER breast tumors, and ment of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania. amplification of TLK2 appears to be enriched in the luminal B 8 Women's Cancer Research Center, University of Pittsburgh, Pitts- breast cancers. The resulting overexpression of TLK2 endows burgh, Pennsylvania. increased invasiveness of luminal breast cancer cells and correlates þ Note: Supplementary data for this article are available at Molecular Cancer with a poorer outcome of ER breast cancer patients. In the current Research Online (http://mcr.aacrjournals.org/). study, we discovered that TLK2 overexpression correlates with Corresponding Author: Xiaosong Wang, University of Pittsburgh, 5117 Centre increased CIN of breast cancers measured by genome-wide copy Avenue, Room G.5a, Pittsburgh, PA 15213. Phone: 412-623-1587; Fax: 412-623- number aberrations, which is independent of the known CIN 1010; E-mail: [email protected] causal factors, such as AURKA and PLK1. TLKs are nuclear- doi: 10.1158/1541-7786.MCR-16-0161 enriched cell-cycle kinases that have maximal activity during S 2016 American Association for Cancer Research. phase and are rapidly inactivated in response to the DNA damage

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induced by ionizing radiation (IR; refs. 10, 11). As the role of temperature and then incubated with Alexa 488 goat anti-rabbit TLK2 in DNA damage response (DDR) was largely based on the antibody (Invitrogen) for 1 hour at room temperature. Cells were studies focusing on TLK1 (11, 12), the role of TLK2 in DDR is analyzed using FACSCanto II Cell Analyzer (BD Biosciences). poorly understood, and there is no report about its function in CIN in breast cancer. Our further experimental studies revealed Neutral comet assay the crucial role of TLK2 overexpression in impairing G2–M check- Comet assay kit was purchased from TREVIGEN and assay was point signaling, delayed DNA repair, and increased CIN. These followed as per the manufacturer's protocols (https://www.trevi- data further support the rationale to target the cell-cycle kinase gen.com/cat/1/3/0/CometAssay/). Briefly, 1 105 cells were col- TLK2 in the management of more aggressive luminal breast lected and suspended in 500 mL of cold PBS. A total of 20 mL of cell cancers. suspension was mixed with 200 mL of LM agarose, and then 50 mL of the cell mixture was placed to the sample area of slide. After Materials and Methods incubation at 4C in the dark for 30 minutes, slides were immersed in chilled lysis solution overnight at 4C and incubated in the Genomic instability index calculation chilled neutral electrophoresis buffer for 30 minutes. Following the Affymetrix SNP 6.0 array–based CNV data (level 3) were electrophoresis at 21 V for 75 minutes at 4 C, slides were immersed retrieved for 1,083 The Cancer Genome Atlas (TCGA) breast- in DNA precipitation solution for 30 minutes at room temperature invasive carcinoma samples. To extract a set of high-confidence and then in 70% EtOH for 30 minutes at room temperature. After copy number alternations (CNA) we used the segment mean drying the samples, slides were incubated with 2.5 mg/mL of threshold of 0.3 for copy number gain and 0.3 for copy number propidium iodide in the dark for 30 minutes, and then slides loss, as previously reported (13). For a given sample, we calculated were dried at 37 C. Tail moment of each cell was analyzed by the genomic instability index from these CNAs using the follow- Comet assay IV software (Perceptive Instruments Ltd.). ing equation: Total CNA segment lengthðÞ bps GII ¼ no: of CNAs Double thymidine block HG19 chromosome sizeðÞ bps T47D cells were treated with 10 mmol/L thymidine (Thy) for 18 hours, released for 9 hours after washing three times with PBS, and The copy number break point index for each breast tumor was then blocked again with 10 mmol/L Thy for 22 hours. calculated as the sum of the copy number break points of each ¼ chromosome ( total number of copy number segments of each Western blot analysis chromosome 1). The cutoff of TLK2 overexpression was cal- Cells were extracted in RIPA lysis buffer (Sigma-Aldrich), sup- þ culated on the basis of median 1 MAD (median absolute plemented with complete protease inhibitor cocktail tablet deviation). MAD is calculated using the R with default constant (Roche). Following primary antibodies were used for Western ¼ ( 1.4826). PAM50-based clinical subtypes of breast cancer for blot analysis: anti-TLK2 (Bethyl Laboratories), anti-GAPDH TCGA samples were derived from the UCSC Cancer Genome (Santa Cruz Biotechnology). Anti-pChk2 (T68), Chk2, pChk1 Browser (https://genome-cancer.ucsc.edu/). (S317), Chk1, pATM (S1981), ATM, pATR (S428), ATR, cyclin B1, pCdk1 (Y15), pH3 (S10), and pAurora kinases antibodies were Cell culture purchased from Cell Signaling Technology. T47D and MCF10A cells were obtained by Dr. Dean P. Edwards (Department of Molecular and Cellular Biology, Baylor College of Engineering doxycycline-inducible plasmids and stable cell Medicine, Houston, TX 77030, USA) from ATCC included in the lines NCI-ATCC ICBP 45 cell line kit. 293FT cells used for lentivirus From the full-length cDNA of TLK2 (Origene, SC115810), the packaging were purchased from Invitrogen. T47D cells were open reading frame (ORF) was subcloned into an inducible lenti- cultured in RPMI 1640 (Cellgro) with 10% FBS (Thermo Fisher viral pTINDLE vector provided by Dr. Xuewen Pan. This vector fi Scienti c). 293FT cells were cultured in DMEM (Thermo Fisher contains an inducible promoter (pTRE-tight) and a transactivator fi Scienti c) with 10% FBS. MCF10A was cultured as described (rtTA3) in a lentiviral backbone. We also engineered the ORF of previously (14). All cell lines were authenticated by characterized yellow fluorescent protein into the pTINDLE vector as a control. cell line core facility of MD Anderson Cancer Center (Houston, After lentivirus packaging, containing doxycycline-inducible plas- TX) performing short tandem repeat analysis of DNA. mid and infection, the stable lines expressing the TLK2 ORF were selected by treating with Geneticin (Invitrogen). Zero, 100, 200, or g-H2AX and TLK2 immunostaining 2,000 ng/mL of doxycycline was used to express the TLK2 ORF. Cells were prepared on coverslips for immunostaining as described previously (15). For primary antibody, rabbit anti- g-H2AX antibody (Bethyl Laboratories) or mouse anti-g-H2AX Results (Millipore) with rabbit anti-TLK2 (Bethyl Laboratories) were TLK2 overexpression correlates with increased genome-wide used. A total of 500 cells were counted in each condition; >10 copy number aberrations g-H2AX foci–containing cells were considered as positive cells. To examine whether TLK2 overexpression correlates with CIN in breast tumors, we calibrated genome-wide genomic instability FACS analysis of cell cycle and apoptosis index (GII) for all TCGA breast tumor samples profiled by Affyme- For cell-cycle analysis, cells were fixed in 70% ethanol (EtOH) trix SNP 6.0 array. The GII score records the percentage of altered and then stained with propidium iodide (Sigma-Aldrich). For genome and is less affected by the noise of copy number datasets. As mitotic analysis, cells were incubated with rabbit anti-phospho- a result,weobserved a significantpositive correlation between TLK2 H3 antibody (Cell Signaling Technology) for 2 hours at room expression and GII (Spearman correlation: R ¼ 0.393, P < 0.001),

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Figure 1. TLK2 overexpression correlates with chromosomal instability measured by copy number data. A, correlation of the GII and CNB index with TLK2 or TLK1 expression [RNA sequencing (RNS- seq)] in 1,083 invasive breast cancers based on Spearman correlation statistics (copy number and RNA-seq data are from TCGA). B, GII of TCGA breast tumors from different intrinsic breast cancer subtypes classified on the basis of TLK2 expression. P values were calculated on the basis of t test. , P < 0.01; , P < 0.001. The samples categorized by PAM-50 subtypes are further classified as TLK2- overexpressing samples and the "rest" samples (see Materials and Methods for the cutoff of TLK2 overexpression). All intrinsic subtypes show significantly higher genomic instability in samples with TLK2 overexpression.

suggesting the role of TLK2 in the instability of the breast cancer tions (4, 16). The CNB approach will complement the GII genome (Fig. 1A, top). Interestingly, this correlation with GII was approach that does not reﬂect the complex rearrangement not observed for TLK1 expression (Spearman R ¼0.09, P > events underlying these copy number alterations. As a result, 0.9). In addition to GII, we also enumerated the genome-wide this revealed a signiﬁcant association of TLK2 expression with copy number breakpoints (CNB, also known as copy number CNB index (Spearman R ¼ 0.384, P < 0.001), but not TLK1 transitions) for TCGA breast tumors as a quantitative measure expression (Spearman ¼0.067, P > 0.9; Fig. 1A, bottom). of the rearrangement events, leading to copy number aberra- These data support the correlation of TLK2 upregulation with

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increased CIN in breast cancer and its distinct function from changes were assessed by flow cytometry measuring DNA content. TLK1. In both models, a prominently delayed accumulation of G2–M To assess the association of TLK2 overexpression with CIN in phase cells was observed after IR when TLK2 was overexpressed, different breast cancer subtypes, we compared GII index of breast suggesting a possible defect of G2–M arrest. To verify the G2–M tumors classified by intrinsic breast cancer subtypes in the presence checkpoint defect, we synchronized the T47D cells at G1–S border or absence of TLK2 overexpression. Here, the intrinsic subtyping is by double thymidine (DT) block. At 5 hours after release from DT based on the 50-gene PAM50 predictor (see Materials and Meth- block, but before entering G2–M phase (Fig. 3C and Supplemen- ods; ref. 17). The GII indexes are significantly higher in TLK2- tary Fig. S5), cells were irradiated, and mitotic entry was analyzed overexpressing luminal B, luminal A, and basal tumor samples as by phospho-H3 staining (a mitotic biomarker). This revealed a compared with TLK2 low samples (P < 0.001), and to a lesser marked increase in mitotic cells (phospho-H3 percentage) in degree in Her2 subtype samples (P < 0.01; Fig. 1B). This suggests T47D cells overexpressing TLK2 after irradiation, supporting a that the increased GII associated with TLK2 overexpression may G2–M checkpoint defect (Fig. 3C). As G2–M checkpoint defect has not be attributed to the enrichment of TLK2 overexpression in the been linked to increased sensitivity to irradiation and genotoxic luminal B tumors known to harbor increased CIN (1). To assess agents, as seen in the cancer cells treated with Chk1 inhibitors whether TLK2 overexpression correlates with other known drivers (20), we postulate that TLK2 overexpression may have a similar of CIN in breast cancer, such as AURKA and PLK1 (Supplementary effect in breast cancer cells. We thus treated the T47D cells over- Fig. S1; refs. 7, 18), we charted the status of TLK2, AURKA,and expressing TLK2 with irradiation or doxorubicin. Indeed, a mod- PLK1 overexpression together with chromosome instability scores est but significant sensitizing effect was observed for both treat- (Supplementary Fig. S2). The resulting heatmap showed that TLK2 ments with TLK2 overexpression, which supports our reasoning overexpression is independent of AURKA or PLK1 overexpression, (Fig. 4A and B). suggesting TLK2 as an independent factor in promoting CIN in breast cancer (Supplementary Fig. S2A). In addition, the luminal B TLK2 overexpression impairs cell-cycle checkpoint signaling in TLK2, AURKA breast tumors overexpressing all three kinases ( ,and response to DNA damage PLK1 ) show a higher GII compared with the tumors overexpressing Next, we went on to investigate how TLK2 overexpression leads only one or two kinases, and the luminal B tumors that are negative to a G2–M checkpoint defect. The key step to initiate the mitotic for all three kinases showed the lowest average GII level (Supple- processes is activation of the Cdk1/cyclin B complex by dephos- TLK2 mentary Fig. S2B). These observations suggest the role of phorylation of Cdk1 on the inhibitory tyrosine pY15 residue (5), overexpression in promoting CIN of breast cancers. which is directly controlled by the G2–M checkpoint signaling (21). We thus performed Western blot analysis to detect alterations in IR- TLK2 overexpression impairs DNA damage repair process induced G2–M checkpoint signaling in T47D cells overexpressing As TLK kinase activity is quickly inhibited following IR-induced TLK2 (Fig. 4C). Impressively, TLK2 overexpression markedly DDR, we hypothesized that TLK2 amplification and overexpres- repressed the phosphorylation of Chk1 and Chk2 in response to sion may override the DDR signaling, leading to impaired double- IR. In addition, a decrease in pY15 Cdk1 (inactive form) and cyclin strand break (DSB) repair. We thus assessed the DSB repair process B1 level is observed with TLK2 overexpression, followed by IR. induced by gamma irradiation (IR) in T47D and MCF10A cells Cyclin B1 starts to accumulate in G2 phase, whereas dephosphor- inducibly expressing TLK2 using g-H2AX foci formation assay ylation of pY15 Cdk1 and activation of Cdk1 is specifictomitotic g ( -H2AX is a biomarker for DSBs; ref. 19). Interestingly, induction cells (5). As a vast majority of cells with 4n DNA content (G2–M of TLK2 overexpression in T47D or MCF10A cells treated with 2 population) attributes to G2 phase, the total cyclin B1 level will be Gy IR prominently delayed the DSB repair process, leading to an primarily affected by the G2 cell population (22). Thus, the decrease increase of DSBs compared with the controls without TLK2 of pY15 Cdk1 and cyclin B1 implies an increase in the mitotic cell induction, which is sustained for a prolonged period of time population and a decrease in the G2 population following TLK2 (Fig. 2A and Supplementary Fig. S3). This result was corroborated overexpression. Furthermore, consistent with the G2–M checkpoint by the neutral comet assay directly visualizing the DSBs in the defect, TLK2 overexpression leads to increased phosphorylation individual irradiated T47D cells and MCF10A-overexpressing and activation of mitotic kinases, such as Aurora A and increased TLK2 (Fig. 2B). In addition, immunofluorescence staining sug- phospho-H3 (Fig. 4C). Together, these data suggest that over- gests that TLK2 forms nuclear foci in irradiated MCF10A cells, expression of TLK2 may override the DNA damage checkpoint g which partially colocalize with -H2AX foci (Supplementary Fig. signaling via repressing Chk1/2, leading to G2–Mcheckpointdefect S4). This implies the intimate association of TLK2 with DDR. and delayed DSB repair. A schematic illustrating the mechanisms of Moreover, TLK2-driven DSB repair defect seems to be indepen- G2–M checkpoint signaling impaired by TLK2 overexpression is dent of p53 status as TLK2-overexpressing MCF10A (p53 wild shown in Supplementary Fig. S6. type) and T47D (p53 mutant) presented similar DDR alternations (Fig. 2 and Supplementary Fig. S3). Discussion

TLK2 overexpression leads to G2–M checkpoint defect Our genome-wide GII and CNB analysis of TCGA breast tumor Next, we performed cell-cycle analysis to determine whether samples revealed that TLK2 amplification might be one of the T47D and MCF10A cells overexpressing TLK2 have a defect in cell- genetic factors contributing to the outbreak of CIN in the luminal cycle arrest after DNA damage (Fig. 3A and B). The asynchronized B breast tumors. Interestingly, a most recent phosphoproteomics T47D cells inducibly expressing TLK2 were irradiated with 2 Gy IR study of TCGA breast cancers by The Clinical Proteomic Tumor and collected at different time points until 21 hours. The asyn- Analysis Consortium (CPTAC) independently identified TLK2 as chronized MCF10A cells inducibly expressing TLK2 were treated a highly phosphorylated kinase associated with genomic ampli- with different doses of IR and collected after 18 hours. Cell-cycle fications at these loci that preferentially present in luminal breast

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A 120 T47D YFP Dox– 120 MCF10A 100 YFP Dox+ 100 TLK2 Dox– TLK2 Dox– 80 80 TLK2 Dox+ TLK2 Dox+ 60 60 40 40 -H2AX foci -H2AX foci γ γ % Cells with % Cells >10 with % Cells with % Cells >10 with 20 20 0 0 Unt 3 6 9 12 15 18 21 24 (h) Unt 3 6 9 12 15 18 21 24 (h) Time after 2 Gy IR Time after 2 Gy IR

T47D-TLK2 B 16 T47D-TLK2 Unt 15 min 9 h Unt 15 min 9 h Dox– 12 Dox+ *** 8

Tail moment 4

0 – xoD – xoD + Unt 15 min 9 h

MCF10A-TLK2 16 MCF10A-TLK2 Unt 15 min 9 h Unt 15 min 9 h Dox– 12 Dox+ *** 8

4 Tail moment

0 – xoD – xoD + Unt 15 min 9 h

Figure 2. TLK2 overexpression undermines DSB repair. A, TLK2 overexpression in T47D cells (left) or MCF10A (right) cells delays the DSB repair process in response to IR. As a control, yellow fluorescent protein (YFP) was expressed in T47D cells. TLK2 or YFP expression was induced in T47D cells or MCF10A cells by treating 100 ng/mL of doxycycline (Dox) for 48 hours. DSB foci induced by 2 Gy of IR were assessed by g-H2AX staining. The charts show the quantification results counting the percentage of cells with more than 10 g-H2AX foci. The representative microscope images are shown in Supplementary Fig. S3. B, DSBs were visualized by neutral comet assays in T47D (top) and MCF10A cells (bottom) after IR with or without TLK2 induction. TLK2 was overexpressed in T47D or MCF10A cells by treating 100 ng/mL of doxycycline for 48 hours. A total of 8 Gy of IR was applied and cells were incubated for 0, 15 minutes, or 9 hours at 37C. Then, neutral comet assay was performed. Left, representative images; right, quantification results. P values were calculated on the basis of t test. , P < 0.001.

cancer (23). This further supports the importance of TLK2 ampli- show that TLK2 overexpression in T47D cells potently represses fication in luminal tumors. The current study will timely com- the phosphorylation of Chk1 (S317) and Chk2 (T68), which may plement the CPTAC study, revealing the role of TLK2 amplifica- explain the impaired G2–M checkpoint signaling (Fig. 4C and tion in G2–M checkpoint defect and CIN. Our experimental data Supplementary Fig. S6). Finally, our result revealed that TLK2 suggest that ectopic expression of TLK2 in the T47D luminal breast overexpression in T47D cells modestly increases the sensitivity of cancer cells or MCF10A benign breast epithelial leads to delayed breast cancer cells to DNA-damaging agents, such as gamma DNA repair, as evidenced by the g-H2AX foci formation and irradiation and doxorubicin (Fig. 4A and B), consistent with the neutral comet assays. Further cell-cycle analysis after irradiation G2–M checkpoint defect in TLK2-overexpressing cells. Future of T47D or MCF10A cells suggested a G2–M checkpoint defect studies will be required to further examine the effect of TLK2 associated with TLK2 overexpression, which is further verified via overexpression on breast cancer cell sensitivity to DNA-damaging phospho-H3 staining (a mitotic biomarker). These data support agents and the dependence of such an effect on p53 status. In the role of TLK2 in G2–M checkpoint defect and CIN. Presumably, addition, it will be interesting to further examine whether TLK2 such a G2–M checkpoint defect will allow unrepaired DSBs to overexpression induces CIN during tumor initiation process. enter into mitosis, leading to accumulation of DNA CNAs as More importantly, our result revealed the distinct functions described previously (24). In addition, our mechanistic studies of TLK2 from TLK1 in DDR. TLK1 has been considered as a

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A Asynchronous T47D-TLK2 B Asynchronous MCF10A-TLK2

Dox– Dox+ Dox– Dox+

21 (h) 21 (hr after IR) 18 18 8 (Gy) 8 (Gy) IR 15 15 12 12 4 4 9 9 2 2 6 6 Unt Unt 3 3 2n 4n 2n 4n Unt Unt DNA Content DNA Content 2n 4n 2n 4n DNA Content DNA Content Dox– Dox+ Dox– Dox+ 100% 100% 100% 80% 80% 80% 60% 60% G2–M 60% G2–M S S 40% 40% 40% G1 G1 20% 20% 20%

Cell population (%) 0%

Cell population (%) 0% 0% Unt 3 6 9 12 15 18 21 Unt 3 6 9 12 15 18 21 84208420 (Gy) IR (hr after IR) (hr after IR)

C DT Synchronized T47D-TLK2

DT Syn 0 1 7 13 19 25 31 (hr after IR)

Release IR treatment following 5-hr release from DT block

Syn 31 h T47D-TLK2 105 6 ) 4 0.21% 2.11% 2 Dox– 10 5 103 Dox– Dox+ 102 4 0 3 105 0.39% 4.63% 104 2 3 Dox+

10 Phospho-H3 (%) 2 1

Phospho-H3 (log 10 0 0 2n 4n 2n 4n Syn Unt 1 7 13 19 25 31 DNA Content (hr after IR)

Figure 3.

TLK2 overexpression impairs G2–M checkpoint induced by DNA damage. IR-induced cell-cycle changes were assessed by flow cytometry measuring DNA content in T47D and MCF10A cells overexpressing TLK2. TLK2 expression was induced for 24 hours in T47D and MCF10A cells using 200 ng/mL doxycycline. A, T47D cells were then irradiated with 2 Gy IR and collected at indicated times. B, MCF10A cells were treated with 2, 4, or 8 Gy of IR and collected after 18 hours. Cell-cycle changes were assessed by flow cytometry measuring DNA content. C, increased mitotic cell population (phospho-H3 positive) after IR in T47D cells overexpressing TLK2. T47D cells with or without TLK2 overexpression were synchronized at the G1–S border by DT block (10 mmol/L); 5 hours after release, but before entering into G2–M phase (Supplementary Fig. S5A), cells were treated with 8 Gy IR and then collected at indicated times. The percentage of phospho-H3– positive cells was quantified by flow cytometry. Unt, untreated; Syn, cells synchronized at G1–S border by DT block. DT, double thymidine.

guardian of genome integrity (12). As opposed to the CIN (Supplementary Fig. S4), and TLK2 overexpression led to driven by TLK2 overexpression, upregulation of TLK1 led to delayed DNA repair, showing 50% to 100% higher number enhanced DNA repair and increased genomic stability (25). In of cells containing more than 10 g-H2AX foci compared with response to DNA damage, TLK1 localizes to DSBs and functions controlafterIR(Fig.2A).AsTLK2shareslesshomologywith as a molecular chaperone to recruit the Rad9–Hus1–Rad1 (9-1- TLK1 in the non-STK domains (10), it may be possible that 1) complex (25), which initiates an ATR-Chk1–mediated cell- TLK2 may lack the DDR chaperone function possessed by TLK1 cycle checkpoint (26). In our result, overexpressed TLK2 in and thus competitively inhibit the TLK1 function as a DDR MCF10A was also recruited to DNA damage site after IR chaperone after DNA damage. Furthermore, a new study just

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A T47D-TLK2 C DT Synchronized T47D-TLK2 1.2 Dox– Dox+ 1 DT Syn 0 1 7 13 19 25 31 (hr after IR) 0.8 ** 0.6 Release IR treatment following 5-hr 0.4 release from DT block 0.2 Surviving fraction 0 Unt IR (2 Gy) DT Synchronized T47D-TLK2 Dox– Dox+ Syn 1 7 13 19 25 31 Syn 1 7 13 19 25 31 (hr after IR) 100 TLK2 75 Dox– Dox+ Dox– Dox+ pATM (S1981) 250 Unt IR (2 Gy) ATM 250 B 250 pATR (S428)

250 ATR T47D-TLK2 75 pChk2 (T68) 1.2 Dox– 50 Dox+ 75 1 50 Chk2 0.8 50 pChk1 (S317) 0.6 ** Chk1 0.4 50 0.2 Surviving fraction 37 pCdk1 (Y15; 0 inactivated) Unt Doxorubicin (5 nmol/L) 75 50 Cyclin B1 20 pH3 (S10) 15 50 pAurora A 37 pAurora B Dox– Dox+ Dox– Dox+ 37 GAPDH Unt Doxorubicin (5 nmol/L) (kD)

Figure 4. TLK2 overexpression impairs DNA damage checkpoint signaling and modestly increases the sensitivity of T47D luminal breast cancer cells to genotoxic agents. A, and B, TLK2 overexpression in T47D cells led to modest but signiﬁcant increase of cancer cell sensitivity to irradiation and doxorubicin treatment. A total of 100 ng/mL doxycycline was administered for 2 weeks to induce TLK2 overexpression in T47D cells; then, either 2 Gy of IR (A) or 5 nmol/L of doxorubicin (B) was administered for 2 weeks. Clonogenic assays were performed to measure cell viability after IR or doxorubicin treatment. P values were calculated on the basis of t test. , P < 0.01. C, alternations of IR-induced checkpoint signaling after TLK2 overexpression in T47D cells. Western blot analysis was performed using the cell lysates obtained from the same experiment as in Fig. 3C. Unt, untreated; Syn, cells synchronized at G1–S border by DT block. DT, double thymidine.

published during our submission has reported TLK2 as a key Disclosure of Potential Conflicts of Interest regulator of checkpoint recovery from DNA damage–induced No potential conflicts of interest were disclosed. G2 arrest (27). Thus, besides repressing IR-induced Chk1/2 activation, TLK2 overexpression may also contribute to the Authors' Contributions premature mitotic entry via its function in G2 checkpoint Conception and design: J.-A. Kim, R. Schiff, K. Li, X. Wang recovery. Future studies are needed to investigate the interac- Development of methodology: J.-A. Kim, X. Wang tion of the two tousled-like kinases in DDR, and pinpoint the Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J.-A. Kim, J. Veeraraghavan precise mechanisms engaged by TLK2 to repress Chk1/2, and Analysis and interpretation of data (e.g., statistical analysis, biostatistics, – impair G2 Mcheckpoint. computational analysis): J.-A. Kim, M. Anurag, K. Li, X. Wang Together, our data suggest that TLK2 amplification may Writing, review, and/or revision of the manuscript: J.-A. Kim, M. Anurag, contribute to the increased CIN of luminal breast cancer via R. Schiff, K. Li, X. Wang Administrative, technical, or material support (i.e., reporting or organizing impairing the G2–M DNA damage checkpoint. In addition to this observation, our previous study showed that TLK2 over- data, constructing databases): J.-A. Kim Study supervision: R. Schiff, X. Wang expression promotes more aggressive phenotypes in luminal breast cancers and correlates with poor prognosis regardless of Acknowledgments endocrine therapy. Thus, targeting TLK2 may present an attrac- The results published here are in part based upon the data generated by TCGA tive therapeutic strategy for the luminal breast tumors harbor- (dbGaP accession: phs000178.v6.p6). We thank Dr. Dean P. Edwards (Depart- ing TLK2 amplifications with enhanced aggressiveness and ment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, increased CIN. TX) for providing cell lines from the ATCC ICBP45 cell line panel.

926 Mol Cancer Res; 14(10) October 2016 Molecular Cancer Research

Role of TLK2 in Breast Cancer Genomic Instability

Grant Support College of Medicine with funding from the NIH (P30 AI036211, P30 This study was supported by Susan G. Komen foundation PDF12231561 CA125123, and S10 RR024574). (to J.-A. Kim), CDMRP grants W81XWH-12-1-0166 (to X-S. Wang), The costs of publication of this article were defrayed in part by the W81XWH-12-1-0167 (to R. Schiff), W81XWH-13-1-0431 (to J. Veeraragha- payment of page charges. This article must therefore be hereby marked advertisement van), and NIH grants CA181368 (to X-S. Wang), CA183976 (to X-S. Wang), in accordance with 18 U.S.C. Section 1734 solely to indicate and P30-125123-06. The computational infrastructure was supported by this fact. Bayer College of Medicine Dan L. Duncan Cancer Center Biostatistics and Informatics Shared Resource (supported by NCIP30 CA125123). This proj- Received May 12, 2016; revised July 12, 2016; accepted July 20, 2016; ect was also supported by the Cytometry and Cell Sorting Core at the Bayer published OnlineFirst August 3, 2016.

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www.aacrjournals.org Mol Cancer Res; 14(10) October 2016 927

Amplification of TLK2 Induces Genomic Instability via Impairing the G 2−M Checkpoint

Jin-Ah Kim, Meenakshi Anurag, Jamunarani Veeraraghavan, et al.

Mol Cancer Res 2016;14:920-927. Published OnlineFirst August 3, 2016.

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