Fig 2: ConSig-Amp identifies TLK2 as a candidate druggable target frequently amplified in breast cancer.(a) The bioinformatics workflow of ConSig-Amp to discover therapeutically relevant oncogene targets in cancer at genome-wide scale based on copy-number and RNAseq data sets. The ConSig-Amp score is calculated by multiplying the ConSig score (see Methods) with the correlation between gene expression and copy number. (b) Prioritizing amplified breast cancer oncogene targets by ConSig score and Spearman's correlation between copy number (Affymetrix SNP 6.0 array) and gene expression (RNAseq). Data shown here are from TCGA. (c) Representative copy-number data showing amplifications at the TLK2 locus in paired breast tumour and peripheral blood (data from TCGA52), or breast cancer cell lines (data from Heiser et al.21). This figure is based on Affymetrix SNP 6.0 array data annotated with genome build hg18. Positive cell line or tumour samples are sorted based on the level of TLK2 amplifications, and the structures of genes involved in the presented region are shown under the illustration. (d) TLK2 expression (based on RNAseq data) is primarily regulated by gene copy number (based on Affymetrix SNP 6.0 array data). The Spearman's correlation is R=0.81. (e) TLK2 expression in different breast cancer subtypes based on RNAseq data. Copy number and RNAseq expression data shown in d,e are from TCGA. The whiskers indicate the max and min values (excluding outliers) and horizontal lines represent the 1st, 2nd and 3rd quartiles. *P<0.05; ***P<0.001. (f) Kaplan–Meier plots based on multiple gene expression data sets showing correlation of TLK2 overexpression with the outcome of systemically untreated or endocrine-treated ER+ breast cancer patients. HT, hormone treated; Tam, tamoxifen-treated; Unt, untreated. P values are calculated based on log-rank tests.

Fig 3: The therapeutic effect of TLK2 inhibition in a MCF7 preclinical xenograft tumour model.(a) The effect of TLK2 inhibition in the MCF7 xenograft tumours inducibly expressing a TLK2 shRNA, in the presence or absence of concomitant tamoxifen treatment. The average tumour growth in each treatment group (8 mice per group). Error bars represent the s.d. of tumour volumes of 8 mice measurements per condition. P values were calculated based on ANOVA to compare the tumour volumes. (b) Kaplan–Meier survival plot comparing the progression-free survival of different treatment groups (based on tumour-doubling time). Generalized Wilcoxon test was used to calculate the P values for comparing progression-free survival between different treatment groups. (c) Quantitative western blot analysis of TLK2 protein expression in the tumours harvested after 15 days of treatment (5 mice/group), or at the end point (8 mice per group). Error bars represent the s.d. of relative TLK2 levels of 5 or 8 mice measurements per condition. *P<0.05; **P<0.01; ***P<0.001. Dox, doxycycline. Corresponding western blot images are shown in Supplementary Fig. 8.

Fig 4: TLK2-amplified luminal breast cancer cells respond differentially to TLK2 or TLK1 inhibition.(a) Cell cycle profile of MCF7 cells synchronized by nocodazole block after TLK2 or TLK1 knockdown. After TLK2 or TLK1 silencing by transfecting 10 nM of esiTLK2 or siTLK1 for 24 h, MCF7 cells were synchronized at mitosis using 200 nM nocodazole for 15 h, and then released. Cells were collected at the indicated time after cell cycle release. To precisely determine S-phase cell population, 10 μM of BrdU was added for 1.5 h before cell collection. The cell cycle distributions were determined based on DNA content and BrdU incorporation (Supplementary Fig. 12). (b) Western blot was done to examine the changes of key signalling molecules involved in G1/S cell cycle regulation and apoptosis using the cell lysates obtained from same experiment as in Fig. 8a. ‘Noc' indicates the MCF7 cells synchronized at mitosis by nocodazole block (before cell cycle release). (c) Cell apoptosis assessed by Annexin V assay in asynchronized MCF7 and MDAMB361 cells following 20 nM of esiTLK2, siTLK1, or siCtrl treatment for 72 h. Error bars represent the s.d. of two replicate measurements per condition. P values are calculated based on t-test. **P<0.01.

Fig 5: TLK2 inhibition results in impaired G1/S progression and induction of apoptosis in TLK2-amplified luminal breast cancer cells.(a) Flow cytometry results showing cell cycle changes after TLK2 knockdown in asynchronized MCF7 and MDAMB361 cells. 10 nM siRNAs (for MCF7 cells) or indicated concentration of siRNAs (for MDAMB361 cells) were transfected for 72 h. Ctrl, Control. (b) Cell apoptosis assessed by Annexin V assay in asynchronized MCF7 and MDAMB361 cells following TLK2 knockdown via 20 nM esiTLK2 or siTLK2#1 transfection for 72 h. Error bars represent the s.d. of two replicate measurements per condition. P values are calculated based on t-test. *P<0.05; **P<0.01; ***P<0.001. (c) Cell cycle profile of synchronized MCF7 cells (by double thymidine block) after TLK2 inhibition. After TLK2 knockdown for 24 h, MCF7 cells were synchronized at the G1/S border using 2.5 mM double thymidine (DT), and then released. Cells were collected every 2 h after cell cycle release for up to 12 h, and analysed for DNA content using flow cytometry. (d) Western blot was done to examine the changes of key signalling molecules involved in G1/S cell cycle regulation using the cell lysates obtained from the same experiment as in 7c. ‘DT' indicates synchronized MCF7 cells by DT block. (e) A schematic of normal G1/S cell cycle signalling and alternations following TLK2 inhibition (black arrows). In normal cell cycle, the cyclin E level starts to increase in late G1 phase, and then collapses as the cells enter S phase53, followed by increased cyclin A expression5455. Rb regulates G1/S transition by repressing the E2F transcription factors that control the expression of cyclin A. Once Rb is phosphorylated (that is, at S807/S811), it releases E2Fs, which will allow cells to enter S phase5657. p27 inhibits the two G1 cyclin/cdk complexes, cyclin D/Cdk4 and cyclin E/Cdk2 (refs 36, 40), both of which are the key upstream kinases of Rb (ref. 37). During normal G1/S progression, the p27 proteins complexed with G1 cyclin/cdks were phosphorylated by the p27-free cyclin E/Cdk2 complexes at T187, which were then targeted for SKP2-mediated proteasome degradation58. D, Cyclin D. E, Cyclin E. A, Cyclin A. R, restriction point.