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Small Molecule Therapeutics

Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK1/2 Inhibitor for Treatment of Triple-Negative Breast Cancer with a Compromised G1–S Checkpoint

Dan Zhu, Shuichan Xu, Gordafaried Deyanat-Yazdi, Sophie X. Peng, Leo A. Barnes, Rama Krishna Narla, Tam Tran, David Mikolon, Yuhong Ning, Tao Shi, Ning Jiang, Heather K. Raymon, Jennifer R. Riggs and John F. Boylan
DOI: 10.1158/1535-7163.MCT-17-1084 Published August 2018

Abstract

Historically, phenotypic-based drug discovery has yielded a high percentage of novel drugs while uncovering new tumor biology. CC-671 was discovered using a phenotypic screen for compounds that preferentially induced apoptosis in triple-negative breast cancer cell lines while sparing luminal breast cancer cell lines. Detailed in vitro kinase profiling shows CC-671 potently and selectively inhibits two kinases—TTK and CLK2. Cellular mechanism of action studies demonstrate that CC-671 potently inhibits the phosphorylation of KNL1 and SRp75, direct TTK and CLK2 substrates, respectively. Furthermore, CC-671 causes mitotic acceleration and modification of pre-mRNA splicing leading to apoptosis, consistent with cellular TTK and CLK inhibition. Correlative analysis of genomic and potency data against a large panel of breast cancer cell lines identifies breast cancer cells with a dysfunctional G1–S checkpoint as more sensitive to CC-671, suggesting synthetic lethality between G1–S checkpoint and TTK/CLK2 inhibition. Furthermore, significant in vivo CC-671 efficacy was demonstrated in two cell line–derived and one patient tumor-derived xenograft models of triple-negative breast cancer (TNBC) following weekly dosing. These findings are the first to demonstrate the unique inhibitory combination activity of a dual TTK/CLK2 inhibitor that preferably kills TNBC cells and shows synthetic lethality with a compromised G1–S checkpoint in breast cancer cell lines. On the basis of these data, CC-671 was moved forward for clinical development as a potent and selective TTK/CLK2 inhibitor in a subset of patients with TNBC. Mol Cancer Ther; 17(8); 1727–38. ©2018 AACR.

Introduction

Breast cancer is a complex and heterogeneous disease with diverse pathologies, distinct histologic features, and therapeutic responses. Breast cancer is traditionally classified into ER+, HER2+, and ER−PR−HER2− (triple-negative breast cancer, TNBC) subtypes. Although the successful development of targeted therapies in the last decade has significantly improved survival for HER2+ and ER+ breast cancer patient populations, TNBC remains a significant unmet medical need because both traditional cytotoxic and targeted therapies fail to produce robust and lasting clinical benefit. This is likely due to several factors including genetic heterogeneity, rapid evolutionary response to therapies, pathway redundancy, and feedback loops. So far, large-scale sequencing of breast cancer genomes has yielded few actionable genetic alterations in TNBC. Recently, the identification of multiple TNBC subtypes further highlights the challenges of developing targeted therapies in this area (1). We utilized a phenotypic approach to screen for compounds that selectively kill TNBC lines but not ER+ or HER2+ breast cancer lines and identified compounds from the pyrrolopyrimidine chemical class, leading to the discovery of CC-671. CC-671, which potently and selectively inhibits both CDC2-like kinase 2 (CLK2) and TTK enzyme activity, possesses desired in vitro and pharmacokinetic profile, as described in a recent publication (2).

Deregulated alternative splicing generates protein diversity contributing to multiple hallmarks of human malignancy (3). Frequent mutations in splicing machinery have been identified in various cancer types (4). Constitutive and alternative splicing are regulated by serine- and arginine-rich (SR) proteins, a family of non–small nuclear ribonucleoprotein particle (snRNP) splicing factors, which contribute to the oncogenic phenotype (5). Two families of protein kinases, SR-specific protein kinases (SRPK) and CLKs, phosphorylate SR proteins regulating their cellular localization and function (6). CLK family members are constitutively localized in nuclear speckles and show extensive colocalization with SR proteins (7). CLK2, a member of the CLK family, phosphorylates SR proteins as part of a regulatory control mechanism for pre-mRNA processing (8). Recently, CLK2 was shown to act as an oncogene in breast cancer (9). Downregulation of CLK2 resulted in inhibition of breast cancer growth in cell culture and xenograft models (9). Small-molecule inhibition of CLK2 changes the expression of SR protein isoforms and alters splicing in genes involved in growth and survival, providing a strong target rationale (10). TTK, also known as Mps1, is a dual serine/threonine kinase that plays a critical role in spindle assembly checkpoint (SAC) signaling by regulating recruitment of the mitotic checkpoint complex to unattached kinetochores (11, 12). TTK mRNA is upregulated in a variety of tumors, including breast cancer, hepatocellular carcinoma, pancreatic ductal adenocarcinoma, and glioblastoma (13–17). In a breast cancer study, both TTK mRNA and protein overexpression are detected in TNBC cohorts and there is a negative correlation between TTK mRNA expression and poor prognosis in TNBC (13). Moreover, TTK belongs to a signature defined by the top 25 genes overexpressed in chromosome instability (CIN) and aneuploid tumors (18). Recently, mitosis-independent expression of TTK protein was associated with aggressive subgroups and poor survival (15). In addition to its mitosis function, TTK plays an important role in DNA damage repair through phosphorylation of p53, CHK2, and MDM2 (19–21). TTK depletion with siRNA accelerates cell-cycle progression through mitosis and induces cell death in TNBC cell lines (13, 22). PTEN-deficient breast cancer cells are particularly sensitive to siRNA-mediated TTK knockdown (23). These data have focused considerable attention on TTK as potential cancer target. Several small-molecule TTK inhibitors have been described and demonstrated antiproliferative activity in human cancer cells with promising efficacy in preclinical xenograft models (22, 24–32). Particularly, the inhibition of TTK significantly reduced the survival of TNBC cells and showed synergy with docetaxel in cancer cells (15) and a mouse model of human TNBC (33).

Here we show CC-671 impacts mitotic progression and pre-mRNA splicing of genes involved in survival, leading to apoptosis in TNBC cells and tumors. Correlative genomic analysis in a panel of breast cancer cell lines identified breast cancer cells with mutations in either CDKN2A, RB, or TP53 genes as more sensitive to CC-671 inhibition (34). CC-671 also produces potent antiproliferative and proapoptotic effects in a wide range of cancer cell lines. Furthermore, intravenous administration of CC-671 to TNBC tumor bearing mice resulted in significant tumor inhibition at well-tolerated doses. Together, our data provide compelling evidence that selective dual inhibition of CLK2 and TTK represents a promising therapy for patients with TNBC with CDKN2A or RB mutation.

Materials and Methods

Cell lines

Forty-nine breast cancer cell lines were purchased from ATCC and DSMZ and maintained in media recommended by the vendors. Upon receipt, each cell line was expanded, confirmed with no mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza), and frozen down at low passages in liquid nitrogen. Cells with no more than 15 passages were used for all cellular studies. Identity of each cell line was authenticated at the vendors and confirmed by comparing gene expression and copy number variation data with gene expression and SNP data of each cell line from CCLE via unsupervised hierarchical clustering. The luminal and basal subtype classification was based upon public information and verified by gene expression data. The estrogen receptor (ER), progesterone receptor (PR), and HER2 status of each cell line was verified by genomic data. Mutation status of relevant oncogenes and tumor suppressor genes was based upon the Catalog of U.S. Cancer Genome Project (CGP) and Cancer Cell Line Encyclopedia (CCLE) database. Additional information on Oncopanel cell lines can be found in the Supplementary Materials and Methods.

RNA-Seq and RT-PCR

CC-671 was synthesized at Celgene using the experimental methods described in ref. 2. CAL51 cells were treated with 3 or 10 μmol/L CC-671 for 6 or 24 hours. Cells were harvested and RNA was isolated by RNeasy Kit (Qiagen). Poly-A selection, strand-specific RNA library, multiplexing indexed libraries, and sequencing with 2 × 100-bp read lengths, and 150M target reads per sample were performed by SeqWright. The RNA-seq data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE113426. HTSeq package was used to quantify gene expression level for each gene locus and differential gene expression was identified using R package “edgeR.” Genes with FDR adjusted P value <0.05 and absolute fold change ≥2 were considered significant. A coverage filter (≥1 sample has ≥50 read counts) was used to remove low coverage genes. Gene set enrichment analysis was performed to identify pathways impacted by compound treatment. Any pathways with FDR adjusted P value <0.05 were considered significant. Splicing effect by treatment was evaluated with DEXSeq. Exons with FDR adjusted P value <0.05, and absolute fold change ≥2 are considered significant. R package “GSEABase” (hypergeometric test) was used to identify pathways overrepresented by genes whose splicing is changed by compound treatment (FDR adjusted P value <0.05).

Reverse transcription was carried out using Super Scripts II Reverse Transcriptase (Life Technologies). Synthesized cDNA was amplified and run in 2% agarose gel. PCR primer sequences are shown in Supplementary Table S1.

Immunoblot

Cell lysates or homogenized tumor tissue samples were evaluated by SDS-PAGE and immunoblotting using the following primary antibodies: phospho-TTK T686 (custom-made rabbit mAb), p-KNL1T943 (custom-made polyclonal antibody), phospho-SR (mouse 1H4G7; Invitrogen), cyclin B1 (Cell Signaling Technology), securin (Cell Signaling Technology), GAPDH, and β-actin (Sigma). Specifically, p-KNL1T943 polyclonal antibody was raised by immunizing rabbits with peptide MEIpTRSHTTALEC (GenScript). The p-KNL1T943 antibody was validated with cell lysates from nocodazole-arrested mitotic CAL51 cells with or without selective TTK inhibitor treatment.

Array scan analysis

The mitotic population of cancer cells was determined using immunofluorescence analysis of phospho-histone H3 at Serine 10 as mitotic marker. Briefly, cells were plated in 96-well plates (PerkinElmer) and treated with 100 ng/mL of nocodazole for 20 hours. Various concentrations of compound or 0.2% DMSO were added to cells for 4 hours. Fixed and permeabilized cells were then stained with phospho-histone H3 antibody (Millipore). Plates were scanned and read using ArrayScan VTI HCS (Thermo Scientific). ArrayScan software Compartmental Analysis Bio application was used to calculate the percentage of phospho-histone H3-positive cells.

Time lapse

U2OS GFP-TUBA1B cells (Sigma Aldrich) were plated and allowed to attach overnight in 5% CO2 at 37°C. Ten minutes after compound addition, the plate was scanned on Perkin Elmer Operetta High Content Imager using a 20× objective lens on confocal mode in a prewarmed 37°C live cell chamber. Each field of view was imaged every 2.75 minutes for a total length of 4 hours to create time-lapse data. Using Volocity software (Perkin Elmer), U2OS GFP-TUBA1B cells were tracked to determine mitotic length. Mitotic length is measured from when GFP-tubulin intensity rises above 1,500 in a cell as determined by Volocity software, and ends when signal intensity drops below 1,500 in the cell. At least 12 cells were tracked between three fields of view for each condition.

Cellular proliferation and colony formation assay

Compound was spotted via an acoustic dispenser (EDC ATS-100) into an empty 384-well plate. Cells from a panel of 49 breast cancer cell lines were diluted to desired densities and added directly to the compound-spotted plates. At the time of setup (t0), initial cell number was assessed via a viability assay (Cell Titer-Glo) and read for luminescence. After 96 hours, viability was assessed via Cell Titer-Glo (Promega). Each cell line was assayed for growth inhibition by CC-671 for at least two independent tests with duplicates for individual test. All data were normalized and represented as a percentage of the DMSO-treated control cells. Compound sensitivity results were derived from the fitted dose–response curve and either then expressed as an IC50 or AUCR, which is the standard area under the dose–response curve (AUC; ref. 35) normalized to the AUC corresponding to theoretical no inhibition.

For the colony formation assay, CAL51 cells were seeded in 96-well plates (Costar) at 500 cells/well, cultured overnight, and treated with CC-671. Plates were placed in the IncuCyte-FLR instrument (Essen Biosciences) and bright field images were taken every day for 21 days. CC-671 was replaced weekly during this study.

Apoptosis assays

For the caspase-3/7 assay, two hours after compound addition, NucView 488 substrate (Biotium) was added to each well of 96-well plates with CAL51 cells. Plates were placed in the IncuCyte-FLR instrument and fluorescence readings were taken every 2 hours, starting at 2 hours after compound treatment and up to 72 hours. The fold of apoptosis was calculated as the ratio of fluorescence signal between compound and DMSO-treated well. For TUNEL assay, CAL51 cells treated with compound for 48 hours were prepared for flow cytometry following TUNEL and propidium iodide staining (Roche Life Sciences). Cells were analyzed on a FACSCalibur Flow Cytometer (Becton Dickinson) and data were processed using FlowJo software.

In vivo studies

All animal studies performed were approved by the Celgene Institutional Animal Care and Use Committee (IACUC; Supplementary Materials and Methods). CC-671 was prepared in ESPS (5% ethanol/12.5% solutol/25% PEG300 and 57.5% saline). The compound was freshly formulated for each dose in xenograft efficacy (Supplementary Materials and Methods) and mechanism-of-action studies. Docetaxel was purchased from Actavis Pharma, Inc., and prepared according to manufacturer's instructions.

Mechanism-of-action studies.

Mice bearing CAL51, MDA-MB-231, or patient-derived xenograft (PDX) tumors ranging from 300 to 500 mm3 were dosed with vehicle or CC-671 at doses ranging between 10 and 40 mg/kg. At predetermined time points after the dose of vehicle or CC-671, the mice were euthanized and the tumors were dissected and processed for Western blot analysis, TUNEL, ELISA, IHC, or Mesoscale assays, per the manufacturer's instructions.

IHC.

Tumors fixed in 10% neutral-buffered formalin were used for histology and IHC. Standard IHC assay using anti-p-KNL1T943 antibody at 0.4 μg/mL was applied to 5- to 10-μm-thick deparaffinized sections. Phosphatase treatment was then performed to confirm phospho-specific signal detected by the antibody. Mitotic CAL51 cell pellets positive control and secondary only negative control were included in each assay.

Results

CC-671 is a potent and selective inhibitor of CLK2 and TTK

A series of potent and selective TTK and CLK2 inhibitors were initially identified from a phenotypic screen for compounds that selectively inhibit the proliferation of TNBC over luminal breast cancer lines. The SAR was driven with a set of breast cancer cell lines (Table 1) and compounds were prioritized on the basis of their selective inhibition of TNBC cell proliferation. A compound (CC-671) with potent and selective TNBC cell activity (Table 1) and acceptable pharmacokinetic and toxicity profiles was selected for further evaluation (Fig. 1; ref. 2). On the basis of the compound structures, we hypothesized that the TNBC antiproliferative activity could be due to kinase binding and inhibition of the pyrrolopyrimidine core to the kinase hinge active site region. Invitrogen kinase panel profiling results show that seven kinases in this panel were inhibited by greater than 80% upon treatment of CC-671 at 3 μmol/L (Supplementary Table S2). Subsequently, CC-671 potency for the seven kinases was determined, showing CC-671 as a potent CLK2 inhibitor with an IC50 value of 6.3 nmol/L (Table 2). CC-671 was also tested in the LanthaScreen TTK binding assay giving an IC50 value of 5.0 nmol/L, as shown in Table 2.

View this table:
Table 1.

Cellular potency of CC-671 in TNBC and luminal breast cancer cell lines

Figure 1.
Figure 1.

Structure of CC-671.

View this table:
Table 2.

IC50 values of the seven cross-reacting kinases identified from kinase panel and TTK

CLK and SRPK phosphorylate SR proteins to regulate cellular pre-mRNA splicing (6). We first confirmed CLK2 as the main kinase for SRp75 phosphorylation in CAL51 cells with siRNA approach. Knockdown of CLK2 by siRNA in CAL51 cells resulted in significant decrease of phosphorylated SRp75 (Supplementary Fig. S1). Because CC-671 did not inhibit either SRPK family members (Supplementary Table S2) or CLK1 potently (Table 2), the status of phospho-SR proteins could be used to evaluate CC-671 inhibition of CLK2 activity. CC-671 treatment led to concentration-dependent reduction of phosphorylated SRp75 and SRp55, as shown in Fig. 2A. The concentration of CC-671 that inhibited cellular SR protein phosphorylation by 50% is 549 nmol/L. Inhibition of pSRp75 and pSRp55 in vivo was determined in CAL51 tumors treated with CC-671. Single-dose intravenous administration of 20 mg/kg CC-671 caused reduction of phosphorylated SRp75 and SRp55 in CAL51 tumors (Fig. 2B). To further characterize CC-671 as a CLK2 inhibitor, RNA-seq analyses was performed in compound-treated CAL51 cells to monitor global alternative splicing patterns. A total of 16 samples were included in this study, including four treatment groups with three biological replicates and two vehicle control groups with two biological replicates. Differential exon usage analysis demonstrated that CC-671 changed alternative splicing of many genes (Fig. 2C). In addition, different sets of genes are impacted by CC-671 at both the alternative splicing and mRNA expression (Fig. 2D). Genes impacted by alternative splicing shared a set of common pathways with genes altered by mRNA expression (Fig. 2D). This indicates that CC-671 regulates transcription via both gene expression and alternative splicing mechanisms. We further confirmed changes in specific alternative splicing events with RT-PCR. Interestingly, PARPBP, a gene that is subject to alternative splicing regulation by SRSF1 in lung cancer cell lines (36), was one of the genes that showed the most significant changes in splice variants (Fig. 2F). RT-PCR result confirmed exon 2 usage of PARPBP was increased in CAL51 cells upon CC-671 treatment (Fig. 2G). CC-671 treatment led to decrease of exon 3 usage in USP14 (Fig. 2G and H), which serves as a positive regulator of the Wnt signaling pathway (37). The antiapoptotic isoform of Bcl-2 (Bcl-2L) was significantly decreased with no change in the Bcl-2S isoform (Fig. 2E and H).

Figure 2.
Figure 2.

CC-671 potently inhibits phosphorylation of CLK2 substrates and alters alternative splicing pattern of genes involved in survival and Wnt signaling. A, CC-671 inhibits phosphorylation of CLK2 substrates in cells. CAL51 cells were treated with different concentrations of CC-671 for 2 hours. Proteins were extracted from treated cells and analyzed by Western blot analysis using antibody against p-SR proteins (1H4 mAb). B, CC-671 potently inhibits phosphorylation of CLK2 substrates in tumors. Animals with CAL51 tumors were treated with a single dose of CC-671 (20 mg/kg), and tumors were collected at 4 and 24 hours postdose. Proteins were extracted from the tumors and analyzed by Western blot analysis using antibody against p-SR proteins. Each data point represents the mean of each time point. Error bars, SEM. P values were calculated by using one-way ANOVA with Dunnett post hoc analysis. C, CC-671 impacts exon usage of many genes. The number of exons passed the filtering criteria (adj. P < 0.05, fold change ≥2) for each treatment are plotted, red = increased exon usage by treatment, blue = decreased exon usage by treatment, gray = number of genes represented by exons. D, CC-671 alters gene expression and alternative splicing. Genes whose mRNA expression level significantly changed by 3 μmol/L CC-671 are compared with genes whose splicing are significantly changed by the same compound treatment. Significance cutoff for both sets are the same: adj. P< 0.05, fold change ≥2. Enriched pathways by genes whose expression affected by compound and whose splicing affected by compound are also compared. Majority of pathways enriched by both sets are common. E, Exon map of Bcl-2 indicates the decreased usage of exon 2 upon compound treatment. F, Exon map of PARPBP indicates the increased usage of exon 2 upon compound treatment at both 6- and 24-hour time points. G, Exon map of USP14 indicates the decreased usage of exon 3 upon 6 hours compound treatment. H, RT-PCR analyses of Bcl-2, PARPBP, and USP14 confirmed the RNA-seq data.

TTK was previously reported to be heavily phosphorylated during mitosis and autophosphorylation at T686 is associated with the active kinase (38). A rabbit mAb against TTK phospho-T686 was generated. To determine whether CC-671 has a direct impact on TTK autophosphorylation, lysates of nocodazole-arrested cancer cells were harvested and analyzed following 1-hour compound treatment (Fig. 3A). Notably, CC-671 caused a complete TTK dephosphorylation at T686 in CAL51 cells at submicromolar concentrations. The potencies for CC-671 inhibition of TTK autophosphorylation were also determined in MDA-MB-231 and MDA-MB-468 cells. CC-671 inhibited TTK autophosphorylation in all three TNBC cell lines with IC50 in the range of 50 to 150 nmol/L.

Figure 3.
Figure 3.

CC-671 blocks phosphorylation of TTK substrates and accelerates mitotic exit in vitro and in vivo. A, Western blot analysis of KNL1 Thr943 phosphorylation, TTK autophosphorylation, total level of cyclin B1, and securin in nocodazole-arrested CAL51 cells treated with different concentrations of CC-671 for 1 hour. B, ArrayScan analysis of histone H3 Ser10 phosphorylation in nocodazole-arrested CAL51 cells treated with different concentrations of CC-671 for 2 hours. The percentage of cells with positive p-HH3 Ser10 staining was calculated. C, Time-lapse of GFP-α-tubulin–expressing U2OS cells. Images were taken every 2.75 minutes for 4 hours. Mitotic length was calculated as the mean of 12 mitotic cells treated by DMSO or CC-671 at different concentrations as labeled. D, Decrease of p-KNL1T943 (brown staining) with a single dose of CC-671 (10 mg/kg) in AA0851L TNBC PDX tumors. Tumor-bearing animals were treated with a single dose of CC-671, and tumors were processed for IHC of p-KNL1T943. E, Significant decrease of pHH3 (Ser10) with a single dose of CC-671 in AA0851L TNBC PDX (20 mg/kg, a) and MDA-MB-231 (40 mg/kg, b) tumors. The tumor lysates were processed for the analysis of pHH3 (Ser10) using MesoScale (a) or Western blot analysis (b). Each data point represents the mean of each time point. Error bars, SEM. P values were calculated by using one-way ANOVA with Dunnett post hoc analysis.

KNL1 was identified as a biologically relevant substrate of TTK (39). Phosphorylation of KNL1 at multiple methionine–glutamine–leucine–threonine (MELT) motifs by TTK creates docking sites for Bub1/BubR1 recruitment (40, 41). An antibody against phospho-KNL1 at T943 was developed and used to determine the impact of CC-671 on KNL phosphorylation. CC-671 demonstrated inhibition of KNL1 phosphorylation at a potency similar to the TTK autophosphorylation inhibition (Fig. 3A). To determine the activity of CC-671 on TTK substrate KNL1 in vivo, animals with TNBC PDX tumors (AA0851L) were treated with CC-671 and phospho-KNL1 were analyzed with IHC. Single-dose administration of CC-671 (10 mg/kg) caused inhibition of phospho-KNL1 in tumor tissues (Fig. 3D). Kinetic studies demonstrated that CC-671 inhibits phospho-KNL1 in a time-dependent fashion through 48 hours. Significant inhibition persisted between 3 and 24 hours (Fig. 3D). Thereafter, phospho-KNL1 level appears to come back at 48 hours.

CC-671 treatment also led to decreased expression of cyclin B and securin proteins, consistent with TTK inhibition (Fig. 3A). In addition, the percentage of mitotic cells in nocodazole-arrested CAL51 cells was substantially reduced by CC-671 in a concentration-dependent manner, shown by the loss of phospho-histone H3 (pHH3) Ser10–positive cells (Fig. 3B). We also conducted time-lapse microscopy experiments in the human U2OS cell line in which the genomic TUBA1B gene has been tagged with a GFP gene. As expected, TTK inhibition by CC-671 resulted in mitotic acceleration. Mitotic phase length was reduced approximately by half (Fig. 3C). Furthermore, administration of CC-671 significantly reduced pHH3 (Ser 10) in TNBC PDX and MDA-MB-231 (Fig. 3E) tumor models. The greatest inhibition of pHH3 (Ser10) was observed at 1 hour (earliest time point tested; 38%, P < 0.001 compared with the vehicle control) and this inhibition persisted through 24 hours (22%, P < 0.05 compared with the vehicle control). Similar significant inhibition (P < 0.05–0.01) of pHH3 (Ser10) through 24 hours postdose was also observed in MDA-MB-231 tumors (Fig. 3E).

CC-671 induces apoptosis and blocks colony formation of TNBC cells

Two types of apoptosis assays were performed to determine whether CC-671 induced apoptosis in CAL51 cells. First, a caspase 3 substrate was used to detect caspase 3/7 activity in intact cells. As shown in Fig. 4A, CC-671 increased caspase 3/7 activity in a concentration- and time-dependent manner. Caspase-dependent apoptosis was observed as early as 24 hours following compound addition, with induction levels highest around 60 hours (Fig. 4A). Treatment with 30 nmol/L CC-671 was sufficient to induce significant caspase 3/7 activation in CAL51 cells. To understand the dynamics of cell death induced by CC-671 in conjunction with those of the cell cycle, we used a terminal transferase (TUNEL) assay together with propidium iodide (PI) for identification of cell cycle. The data shown in Fig. 4B confirmed that CC-671 treatment led to cell death. These data also indicated that CAL51 cells became apoptotic during G1, S, or G2–M phases. The induction of apoptosis led to elimination of viable colony formation of CAL51 treated by CC-671 at concentrations ≥100 nmol/L in colony formation assay (Fig. 4C). Induction of apoptosis was also observed in vivo with CC-671 in TNBC PDX and MDA-MB-231 tumors. Time course studies with the MDA-MB-231 model show significant induction of cleaved PARP by a single dose of CC-671 (40 mg/kg) persisting out to day 7 (Supplementary Fig. S2; Fig. 4D, a–d). In the TNBC PDX model, there was a significant increase (2- to 3-fold, P < 0.001) in cleaved caspase at 24 and 48 hours (last time point tested) after single dose of CC-671 at 20 mg/kg (Fig. 4D, e).

Figure 4.
Figure 4.

CC-671 induces apoptosis in TNBC cells and tumors and blocks colony formation of TNBC cells in concentration-dependent manner. A, CC-671 induced CAL51 apoptosis over time, as shown by cleaved caspase 3/7. DMSO-treated cells served as negative controls. B, CC-671 induced apoptosis of cells in all cell-cycle phases, as shown by FACS analysis of CAL51 cells treated with CC-671 for 48 hours. Cells were fixed and stained with TUNEL and PI, for apoptosis and cell-cycle markers, respectively. The percentage shown represents the relative difference in TUNEL-positive threshold area between samples from CC-671-treated and vehicle-treated control cells. C, CC-671 treatment at concentrations ≥100 nmol/L eliminates viable colony formation in CAL51 long-term colony formation assay. D, Induction of apoptosis in vivo with a single dose of CC-671 in MDA-MB-231 (a-d) and AA0851L TNBC PDX (e) tumors. a–d, Low (a and b) and high (c and d) magnification of IHC images of untreated (a and c) and CC-671 (40 mg/kg, single dose, day 7)-treated (b and d) tumors showing increased cleaved PARP (brown staining) in the tumors treated with CC-671. e, Time-dependent increase in cleaved caspase in AA0851L TNBC PDX tumors treated with vehicle or CC-671 (20 mg/kg, single dose). Each data point represents the mean of each time point. Error bars represent standard error of the mean P values were calculated by using one-way ANOVA with Dunnett post hoc analysis.

CC-671 demonstrates significant dose-dependent efficacy in TNBC xenograft models

Next, we tested the antitumor activity of CC-671 in Taxotere insensitive (CAL51) and sensitive (MDA-MB-231) TNBC xenograft models. Taxotere insensitivity was defined as <60% tumor inhibition at MTD (>15% body weight loss). Weekly intravenous administration was chosen based on improved tolerability with equivalent efficacy as compared with a more frequent dosing paradigm (every 3 days; ref. 2). Antitumor activity with CC-671 was observed in both models without causing significant body weight loss. In the CAL51 model, there was a 70% reduction in tumor volume at 20 mg/kg compared with the vehicle control (Supplementary Fig. S3A). Docetaxol showed slightly less efficacy compared with CC-671 (Supplementary Fig. S3A) while causing severe body weight loss (>20%; Supplementary Fig. S3B). In the Taxotere-sensitive TNBC model (MDA-MB-231), CC-671 demonstrated an approximate 64% reduction in tumor volume at 20 mg/kg (Supplementary Fig. S3C) with no significant weight loss (Supplementary Fig. S3D). In vivo efficacy of CC-671 (10–40 mg/kg, every 7 days) was further evaluated in TNBC PDX model. As shown in Fig. 5A, there was a dose-dependent tumor inhibition with CC-671 in AA0851L TNBC PDX model. Tumor regression (21%–43%) was also observed at 40 mg/kg with no significant body weight loss (Fig. 5B and C).

Figure 5.
Figure 5.

Antitumor activity of CC-671 in AA0851L TNBC PDX model. Groups of (n = 8 per each treatment group) mice with approximately 200 mm3 AA0851L TNBC PDX tumors were treated intravenously with vehicle, CC-671 (every 7 days), or docetaxol (every 4 days). A, Dose-dependent and statistically significant (P < 0.0001 for all dose levels) tumor inhibition was observed with CC-671 when compared with vehicle in AA0851L TNBC PDX model. Each data point represents the mean tumor volume of each treatment group. Error bars represent standard error of the mean. P values were calculated by using one-way ANOVA with Dunnett post hoc analysis. B and C, CC-671 treatment led to tumor regression (B) with minimum body weight loss (C).

Identification of patient enrichment strategy for CC-671

To confirm the selectivity of CC-671 for TNBC and identify biomarkers for patient selection in breast cancer, the antiproliferative activity of CC-671 was determined across a panel of 49 breast cancer cell lines. CC-671 inhibits the growth of breast cancer cell lines with IC50 values <100 nmol/L in 14 lines and >10 μmol/L in 21 lines (Fig. 6A; Supplementary Table S3). TNBC cell lines are significantly more sensitive to CC-671 than non-TNBC lines (Fig. 6B). Correlation analysis with genomic data identified CDKN2A, RB, and, to less extent, TP53 mutations to be associated with CC-671 sensitivity in breast cancer panel (Fig. 6C). Twelve of 16 of the most sensitive breast cancer lines have either CDKN2A or RB mutations, whereas only one in 16 of the most resistant breast cancer lines have mutations in either gene. Gene expression analysis further confirms the observation. Herschkowitz and colleagues identified 20 genes that are regulated by the RB pathway and associated with proliferation (42). Fig. 6D shows a heatmap of hierarchical clustering of the 20 RB-regulated genes in the 49 breast cancer lines. A combination of low expression of the 20 RB-regulated genes and no mutation in CDKN2A and RB suggests cell lines in cluster A have intact G1–S checkpoint. These cell lines are non-TNBC lines and resistant to CC-671. However, cell lines in cluster B have high expression of RB-regulated genes and are more sensitive to CC-671. Many of them have a mutation in either CDKN2A or RB, suggesting the mutations of CDKN2A and RB likely lead to loss of G1–S checkpoint control and upregulation of E2F-targeted genes that are normally suppressed by RB. Interestingly, several cell lines in cluster B have high expression of RB/proliferation genes despite a lack of mutations in CDKN2A and RB genes, suggesting the existence of other mechanisms contributing to the loss of G1–S checkpoints. In fact, promoter hypermethylation of p16, one of the genes encoded by CDKN2A, has been reported to occur frequently in human tumors (43). TTK is one the 20 genes that are regulated by RB/E2F. TNBC expresses basal type gene signature and breast tumors of basal-like subtype have been shown to have high growth rate and highly express a proliferation signature (41). Low RB expression and loss of heterozygosity of RB locus are associated with high proliferation rate and elevated TTK expression in TNBC/basal-type. Consistently, TNBC lines tend to have high expression of TTK and sensitivity to CC-671 significantly correlate with TTK expression in the breast cancer cell line panel (Fig. 6E). Not surprisingly, TTK expression level correlates with growth rates of cell lines in both the breast cancer panel and the oncopanel (Supplementary Fig. S6A and S6C). CC-671 potency also correlates with growth rates of cancer cell lines in both panels (Supplementary Fig. S6B and S6D).

Figure 6.
Figure 6.

TNBC lines are more sensitive to CC-671 and CDKN2A, RB, and TP53 mutation, RB signature, and TTK expression are associated with sensitivity. A, Antiproliferative activity of CC-671 across 49 breast cancer cell lines. B, Box plots showing correlation of CC-671 potency with molecular subtypes of breast cancer. The molecular subtypes for ER+, HER2+, and triple-negative groups were defined as follows: the ER+ group contains cell lines that are ER+, PR+, or PR−, and HER2−. The HER2+ group contains cell lines that are ER+ or ER−, PR+ or PR−, and HER2+. The triple-negative group contains cell lines that are ER−, PR−, and HER2. C, Box plots showing correlation of CC-671 potency with mutation status of CDKN2A, RB, and TP53. AUCR, area under the curve ratio; CDKN2A, gene encoding cyclin-dependent kinase inhibitors p16 (INK4) and p14 (ARF); mut, mutant; RB1, retinoblastoma protein 1; res, resistant; sens, sensitive; TN, triple-negative breast cancer; TP53, gene encoding tumor suppressor protein p53; TTK, TTK protein kinase (or Mps1); WT, wild type. D, Heatmap of a hierarchical clustering of 20 RB-regulated genes in the 49-breast cancer cell line panel. Top bars indicate mutation status for CDKN2A and RB, ER, PR, HER2 status, and sensitivity and resistance of cell lines to CC-671. The gene-level expression values, which were the means of all corresponding probe sets, were used. The cell lines and genes are arranged by hierarchical clustering. In the colored side column bar, red = up, green = down, and arrow = TTK. E, Box plots showing correlation of CC-671 potency with TTK mRNA in the breast cancer panel. The TNBC lines are shown in red. Non-TNBC lines are shown in gray.

Furthermore, cellular potency of CC-671 was evaluated across a broad panel of 240 cancer cell lines representing various anatomic origins (Supplementary Table S4). The effect of the compound on cell proliferation, apoptosis, and cell-cycle progression was assessed using an image-based, multiplex cell assay (Supplementary Materials and Methods). CC-671 has potent antiproliferative activity against cancer cell lines of different tumor types with IC50 values ranging from 0.03 to 10 μmol/L (Supplementary Fig. S4A). The majority (149/240) of the cell lines were inhibited at IC50 values less than 1 μmol/L, whereas 45 lines showed no response at a concentration of 10 μmol/L demonstrating CC-671 cellular selectivity. In addition, CC-671 potently induces apoptosis in cancer cell lines across many tumor types (Supplementary Table S4; Supplementary Fig. S4B). Cell-cycle analysis using pHH3 Ser10 indicates that CC-671 causes ≥2-fold decrease of pHH3 Ser10 staining in 120 cell lines at a concentration <1 μmol/L, suggesting CC-671 accelerated mitotic exit in these lines (Supplementary Table S4). In general, cell lines representing leukemia, lymphoma, colorectal cancer, head and neck (H&N), and bladder cancers were more sensitive to CC-671 than cell lines from other tumor types. No single mutation has been identified that statistically significantly correlates with sensitivity to CC-671 across the panel. Zaman and colleagues reported that TTK inhibitor NTRC 0066-0 is more potent in inhibiting the proliferation of six cell lines containing active CTNNB1 mutation than the cell lines with wild-type CTNNB1 (32). Ten of the 240 cell lines tested here harbor clinically relevant CTNNB1 mutation in exon 3 and tend to be more sensitive to CC-671 but the correlation did not reach statistical significance (Supplementary Tables S5 and S6; Supplementary Fig. S5). CDKN2A mutation is associated with CC-671 sensitivity in lung cancer and melanoma, whereas TP53 mutation associates with CC-671 resistance in lung cancer (Supplementary Table S7). However, no significant correlation is observed between CDKN2A or RB mutation and CC-671 sensitivity in the 16 breast cancer lines of Oncopanel, presumably due to the small sample size.

Discussion

Resistance to chemotherapeutic agents contributes to a dismal prognosis for patients with TNBC. To seek new therapeutic opportunities for this patient population, we deployed a phenotypic-based approach and discovered CC-671, which preferentially induced apoptosis in TNBC cell lines while sparing luminal breast cancer cell lines. CLK2 and TTK were identified as the main CC-671 targets via kinase profiling. CC-671 demonstrated potent inhibition of CLK2 kinase activity with a mean IC50 of 549 nmol/L in asynchronized CAL51 cells. This compound also potently inhibited cellular TTK kinase activity with a mean IC50 of 49 nmol/L in mitotic CAL51 cells.

In agreement with the important role of CLK2 in regulating alternative splicing, CC-671 induced large-scale splicing alterations. Exon usage of 500 to 1,500 unique genes are significantly affected by CC-671 treatment. Alternative splicing of several genes involved in stemness and survival signaling, such as Bcl-2, PARPBP, and USP14, were further evaluated. Our results showed that specific exon usage in these three genes was significantly altered by CC-671 treatment. Those data are consistent with CC-671 regulating pre-mRNA splicing as a CLK2 inhibitor. Therefore, the CC-671 CLK2 inhibitory activity contributes to alterations of specific splicing events that encode proteins involved in cell survival and stemness.

In the last decade, several mitotic kinases have been identified as potential oncology therapeutic targets. TTK, as a core component of the spindle checkpoint, has garnered intense drug discovery interest. CC-671, a potent TTK inhibitor, dose dependently blocked autophosphorylation of TTK and phosphorylation of KNL1 in nocodazole-arrested cells. CC-671 treatment reduced the duration of mitosis and induced caspase-dependent apoptosis in a concentration- and time-dependent manner. No viable colonies were detected in CAL51 cells treated with CC-671 at concentrations potently inhibiting TTK. The effect of CC-671 on cell cycle was further confirmed in a large panel of cancer cell lines. Among 240 cancer cell lines, CC-671 accelerated mitotic exit in 120 lines, resulting in apoptosis induction and inhibition of proliferation. Importantly, CC-671 has no activity in 45 cancer cell lines, indicating that it is not a general cytotoxic agent. Cell lines representing leukemia, lymphoma, colorectal cancer, head and neck, and bladder cancers were uniformly sensitive to CC-671, whereas cell lines from other tumor types such as breast and lung cancers were differentially sensitive to CC-671.

CC-671 causes potent yet selective growth inhibition and apoptosis induction in many cancer cell lines. In vivo, CC-671 treatment inhibits tumor growth in TNBC xenograft and PDX models at tolerable doses, with no body weight loss. CC-671 inhibits RNA splicing and accelerates mitotic progression at concentration similar to those that produced antiproliferative and apoptosis induction activity in TNBC cells and tumors. These data suggest that CC-671 exhibited the pharmacological activity expected for a potent TTK/CLK2 inhibitor. Recently, TTK mutations in the adenosine triphosphate-binding pocket were identified in cancer cells that are resistant to several TTK inhibitors (30, 44). A critical next step will be to determine whether CC-671 maintains potency in the cancer cells harboring TTK mutants.

To identify biomarkers for patient selection, the antiproliferative potency of CC-671 was determined in a panel of 49 breast cancer cell lines in vitro. TNBC lines in this panel of breast cancer cell lines were significantly more sensitive than non-TNBC lines, confirming TNBC selectivity. Correlation analysis identified mutations in CDKN2A and, to a lesser extent, RB and TP53, as significantly associated with breast cancer cell line sensitivity to CC-671. CDKN2A, a tumor suppressor gene that is frequently mutated in human cancers, encodes both p16INK4a and p14ARF. p16INK4a and p14ARF regulate RB and p53, respectively, and constitute the G1–S checkpoint. A mutation in CDKN2A, RB, or TP53 results in compromised G1–S checkpoint and deregulation of entry into the cell cycle (45). The fact that mutations in three critical genes comprising the G1–S checkpoint all correlate with sensitivity to CC-671 argues that cell lines with dysfunctional G1–S checkpoints are substantially more sensitive to CC-671 treatment than cell lines with functional G1–S checkpoints. CC-671 is a potent inhibitor of TTK, an essential component of the mitotic spindle checkpoint. TTK itself is also a target gene of transcription factor E2F and regulated by RB (42). Mutations in CDKN2A or RB leads to activation of E2F, which upregulates genes for DNA synthesis and mitotic spindle checkpoint genes, such as TTK. Mitotic spindle checkpoint serves as a surveillance system to block aberrant mitosis and ensures that mitosis progresses normally. Our data suggest inhibition of TTK is synthetically lethal with loss of a functional G1–S checkpoint pointing to patients with cancers that have lost G1–S checkpoint being sensitive to a TTK inhibitor such as CC-671.

In summary, the CLK2 and TTK inhibitor CC-671 showed strong growth inhibition and apoptosis induction in TNBC and in other cancer cells with significant efficacy in TNBC tumor models. Genetic and cellular heterogeneity in conjunction with TNBC plasticity conspire to limit the therapeutic effectiveness of targeted therapies. These fundamental elements of tumor biology require orthogonal therapy approaches combining multiple drugs with independent targets. Compared with other published TTK inhibitors, CC-671 has a unique bi-specific profile, targeting TTK and CLK2. Although the CC-671 activity in cancer cells might be mainly attributable to TTK inhibitory activity, the contribution of CC-671 CLK2 inhibition to apoptosis induction and cancer stem cell population warrants further investigation. Overall, CC-671 not only serves as valuable tool for understanding the biological function and synthetic lethality of the CLK2 and TTK inhibitory combination in cells but also represents a new class of TNBC therapy in a defined patient population.

Disclosure of Potential Conflicts of Interest

The authors declare no competing financial interest. All authors are employees of Celgene, except Gordafaried Deyanat-Yazdi, Ning Jiang, and Yuhong Ning, who were Celgene employees at the time of their contribution to this work.

Authors' Contributions

Conception and design: D. Zhu, S. Xu, G. Deyanat-Yazdi, R.K. Narla, T. Tran, J.R. Riggs, J.F. Boylan

Development of methodology: D. Zhu, G. Deyanat-Yazdi, R.K. Narla, T. Tran, D. Mikolon

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D. Zhu, G. Deyanat-Yazdi, S.X. Peng, L.A. Barnes, R.K. Narla, T. Tran, D. Mikolon, N. Jiang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D. Zhu, S. Xu, G. Deyanat-Yazdi, L.A. Barnes, R.K. Narla, T. Tran, D. Mikolon, Y. Ning, T. Shi, N. Jiang, H.K. Raymon, J.R. Riggs, J.F. Boylan

Writing, review, and/or revision of the manuscript: D. Zhu, S. Xu, R.K. Narla, T. Tran, D. Mikolon, Y. Ning, T. Shi, H.K. Raymon, J.R. Riggs, J.F. Boylan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Zhu, R.K. Narla, T. Tran, D. Mikolon, J.R. Riggs

Study supervision: D. Zhu, S. Xu, R.K. Narla, T. Tran, H.K. Raymon, J.F. Boylan

Acknowledgments

The authors thank the Celgene San Diego DMPK, Compound Management, and Histology Lab for project support. In addition, compound resupply was provided by the Celgene Chemistry Department, specifically Roy Harris, Paul Erdman, and Mark Nagy. This work was supported by Celgene Corporation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Received November 8, 2017.
  • Revision received March 13, 2018.
  • Accepted May 8, 2018.
  • Published first June 4, 2018.

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Synthetic Lethal Strategy Identifies a Potent and Selective TTK and CLK1/2 Inhibitor for Treatment of Triple-Negative Breast Cancer with a Compromised G1–S Checkpoint
Dan Zhu, Shuichan Xu, Gordafaried Deyanat-Yazdi, Sophie X. Peng, Leo A. Barnes, Rama Krishna Narla, Tam Tran, David Mikolon, Yuhong Ning, Tao Shi, Ning Jiang, Heather K. Raymon, Jennifer R. Riggs and John F. Boylan
Mol Cancer Ther August 1 2018 (17) (8) 1727-1738; DOI: 10.1158/1535-7163.MCT-17-1084
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