CDK7-dependent transcriptional addiction in triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-negative but not hormone receptor-positive breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An "Achilles cluster" of TNBC-specific genes is especially sensitive to CDK7 inhibition and frequently associated with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer.

Figure 1. CDK7 Inhibition Selectively Targets TNBC Cells

A. Cell growth curves of triple-negative (red) and ER/PR+ (blue) breast cancer cell lines. Cells were treated with increasing concentrations of THZ1 for 48 hours. Cell were then fixed and stained for the quantification of cell growth. Data are presented as means ± SD. B. Bright-field images of cells that were treated with vehicle control or THZ1 (33 nM) for 7 days. Note that THZ1 induced cell death in triple-negative but not ER/PR+ breast cancer cells. C. THZ1 inhibits RNAPII CTD phosphorylation in both triple-negative (MDA-MB-468) and ER/PR+ (ZR-75-1) breast cancer cells. Cells were treated with vehicle control (first lane) or increasing concentrations of indicated drug (2, 10, 50, 250, 1250, 6250 nM) for 4 hours before lysates were prepared for immunoblotting. D. Immunoblotting analysis of lysates harvested from cells treated for 24 hours with vehicle control or THZ1 (100 nM). Samples in the order of loading were triple-negative (MDA-MB-468, BT549, HCC1187), ER/PR+ (ZR-75-1, T47D) breast cancer cells, and normal human cells (RPE, BJ1). E. Indicated TNBC (red) or ER/PR+ (blue) primary cultures were treated with increasing concentrations of THZ1. Cells were subjected to CellTiter-Glow Luminescent Cell Viability Assay after 48 hours of treatment. Data were represented as mean ± SD. F. Triple-negative (DFBC12-06) or ER/PR+ (DFBC14-15) primary culture was treated with vehicle control or THZ1 (250 nM) for 24 hours. Cells were subjected to LIVE/DEAD Cell Viability Assay to indicate live (green) and dead (red) cells. G. THZ1 inhibits RNAPII CTD phosphorylation and induces apoptosis in primary TNBC cells. Primary TNBC culture (DFBC12-58) was treated with vehicle control (first lane) or indicated concentrations of THZ1 for 24 hours before lysates were prepared for immunoblotting. See also Figure S1, Movie S1–2.

Figure 2. An Analog of THZ1, and the Effect of CDK7 inhibition on the Growth of Triple-Negative Breast Tumors

A. Structure of THZ1 and THZ2. The groups of 4-acrylamide-benzamide in THZ1 and 3-acrylamide-benzamide in THZ2 are colored red. B. Stability of THZ1 and THZ2 in vivo. Mice were administered by tail vein injection of a single dose of THZ1 or THZ2, and blood samples were collected at different timepoints. Concentrations of THZ1 and THZ2 in plasma samples were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach. C. In vitro IC50 for THZ2’s potency in binding to indicated CDK. The LanthaScreen Eu Kinase Binding assay (Invitrogen) was performed with indicated CDKs and their associated cyclins, in the presence of different concentration of THZ2. The IC50 values indicate the affinity of THZ2 towards the ATP binding pocket of CDK. D. Cell growth curve of breast cancer cells treated with increasing concentrations of THZ2 for 48 hours. Data are presented as mean ± SD. E. Bright-field images of cells treated with vehicle control, or THZ2 (333 nM), or THZ1-R (333 nM) for two days. F. Cell growth curve of indicated TNBC cell lines that were treated with increasing concentrations of THZ2 for 7 days. Upon harvest, cells were fixed, stained with crystal violet followed by extraction of the staining for the quantification of proliferation. Data are presented as mean ± SD G. Growth of triple-negative breast tumors in nude mice treated with vehicle (n=8) or THZ2 (n=7; 10 mg/kg intraperitoneal). Mean ± SEM values are presented, * p < 0.05 (Student’s t-test). H. Immunoblotting of tumor lysates harvested from nude mice treated with vehicle or THZ2 (10 mg/kg intraperitoneal) for 2 days. Tumors were isolated 3 hours after last treatment, and subjected to the preparation of RIPA lysates. Three independent samples from each treatment were loaded in duplicates. I. Growth of patient-derived triple-negative breast tumors (DFBC11-26) in NOD-SCID mice treated with vehicle (n=4) or THZ1 (n=6; 10 mg/kg intraperitoneal). Mean ± SEM values are presented, ** p < 0.01 (Student’s t-test). J. Growth of patient-derived triple-negative breast tumors (DFBC13-11) in NOD-SCID mice treated with vehicle (n=6) or THZ1 (n=5; 10 mg/kg intraperitoneal). Mean ± SEM values are presented, * p < 0.05, ** p < 0.01 (Student’s t-test). K. Hematoxylin and eosin (H&E) staining of tissue sections (DFBC13-11) indicating tumor regression after THZ1 treatment. Note that THZ1-treated tumor shows a loss of cellularity compared to control. Images on the left and right were captured using 4× and 10× object lens respectively. L. Immunoblotting of tumor lysates (DFBC11-26) harvested from mice treated with vehicle or THZ1 (10 mg/kg intraperitoneal) for 21 days. Samples (two and three for vehicle and THZ1 treated groups, respectively) were loaded in duplicates. See also Figure S2, Table S1.

Figure 3. Loss of CDK7 Impairs TNBC Cell Growth and Tumorigenesis

A. Loss of CDK7 in TNBC cells impairs cell viability and proliferation. The left, middle, and right panels show the bright-field images, the crystal violet staining of cells, and the quantification of cell growth, respectively. Data in the right panel are presented as mean ± SD, * p < 0.01, *** p < 0.0001 (Student’s t-test). B. Tumor volume of xenografts derived from cells infected with sg_GFP or sg_CDK7 (sg_CDK7_2 in Figure 3A). Cells were infected with lentivirus, selected with puromycin for two days, and then harvested for transplantation. Two million MDA-MB-468 or MDA-MB-231, or 4 million SUM149 cells (viability > 94% for all groups, assayed by trypan blue exclusion) were transplanted into mammary fat pads of nude mice. Tumor volume was measured 4 weeks after transplantation for the lines of SUM149 and MDA-MB-231, and 5 weeks for MDA-MB-468. Data were represented as mean± SEM, with p value indicated. The right panel shows immunoblotting from cultured cells that were used for transplantation. Note that the protein abundance of CDK7 was efficiently decreased by sg_CDK7. C. Immunoblotting of lysates from cells introduced with CRISPR constructs. Lentivirus-infected and puromycin-selected cells were seed in 6-well plate (20, 000 cells per well), and harvested in four days. RIPA lysates were subjected to the analysis of apoptotic cell death (indicated by PARP and Caspase 3 cleavage). D. Cell cycle analysis of cells infected with lenti-virus encoding sg_GFP, two independent sgRNA targeting CDK7. Cells were prepared as in (C), and then fixed for cell cycle assay. See also Figure S3.

Figure 4. Unique Dependence on CDK7 by TNBC Cells

A. Immunoblotting of lysates from MDA-MB-468 cells that were infected with lentivirus encoding Cas9 and sgRNA targeting GFP or individual transcriptional CDK. * denotes a non-specific signal for anti-CDK13. B. Role of transcriptional CDK for the indicated TNBC cells. After infection and selection with puromycin (1.5 µg/ml, 48 h), cells were seeded in 12-well plate (5000 per well for MDA-MB-468, 10,000 per well for BT549). Cells were fixed after 11 days, and stained with crystal violet. C. Quantification of cell proliferation. Cells were treated as in (B). The staining was subsequently extracted for measurement of absorbance, to quantify cell growth. Data are presented as mean ± SD, * p < 0.0001 (Students’ t-test) D. Bright-field images of cells infected with virus encoding sg_GFP, sg_CDK7, or sg_CDK9. Cells were assayed as in (B), and imaged with an inverted microscope See also Figure S4.

Figure 5. Genes Expressed Differentially in TNBC versus ER/PR+ Breast Cancer Cells and Sensitive to CDK7 Inhibition Indicate Critical Cellular Functions for TNBC Survival

A. THZ1 treatment globally affects steady-state mRNA levels. BT549 and MDA-MB-468 TNBC cells were treated with THZ1 at the indicated concentrations for 6 hrs. Heatmaps display the Log2 fold change in gene expression versus vehicle control for the set of expressed transcripts. B. Genes differentially expressed between TNBC and ER/PR+ breast cancer lines. Individual bars represent the difference in expression in TNBC cells versus ER/PR+ cells for a gene. Genes that were differentially expressed in either of two TNBC cell lines (BT549 and MDA-MB-468) relative to two ER/PR+ lines (ZR-75-1 and T47D) were identified as TNBC–specific (right side of Y-axis). Genes that were differentially expressed in either of two ER/PR+ lines relative to two TNBC breast cancer lines were identified as ER/PR+–specific (left side of Y-axis). Genes whose expression decreased by 1.5-fold or greater upon treatment with THZ1 were colored (blue for TNBC-specific; green for ER/PR+-specific). Log2 fold change between TNBC and ER/PR+ expression is shown along the x-axis at the bottom of the image. C. Enriched Gene Ontology functional categories of TNBC–specific genes sensitive to THZ1 treatment. The top enriched Molecular function GO categories are shown. Individual bars represent the Bonferroni-corrected p-value for enrichment of specific gene ontology categories. Values for TNBC-specific, THZ1-sensitive genes are shown in blue. Values for ER/PR+-specific, THZ1-sensitive genes are shown in green. D. Depiction of signaling pathways and transcription factors that comprise Achilles Cluster genes. Highlighted genes are found in the Achilles cluster. E. Achilles cluster genes are enriched in super-enhancers—associated genes. Venn diagram showing the overlap (66) between the genes that comprise the Achilles cluster (166) and genes that have TNBC super-enhancers (SE) in either MDA-MB-468 or BT549 (1207) (top). Total H3K27Ac ChIP-seq signal (length * density) in enhancer regions for all stitched enhancers in MDA-MB-468 TNBC cell line. Enhancers are ranked by increasing H3K27Ac ChIP-seq signal (bottom). Highlighted super-enhancers are associated with selected members of the Achilles cluster. F. Enrichment of DNA-binding motifs targeted by signaling transcription factors in constituent enhancers of super-enhancers regulating Achilles cluster genes in TNBC cells. G. Genes most strongly downregulated by THZ1 treatment in patient-derived TNBC primary cells are enriched for Achilles cluster genes. Gene set enrichment analysis of Achilles cluster genes in comparison to genes downregulated in TNBC primary cultures (DFBC12-06, DFBC12-58, DFBC13-11) following treatment with THZ1 (250 nM) for 6 h. GSEA-supplied p-values are given. See also Figure S5, Table S2–4.

Figure 6. Functions of Achilles Cluster Genes in TNBC Cells

A. CRIPSR-mediated editing of selected TNBC Achilles cluster genes in TNBC cell line. MDA-MB-468 cells were infected with lentivirus encoding indicated sgRNA, selected with puromycin. (Left) Immunoblotting for the expression of indicated genes. (Right top) Cells were seeded in 12-well plates (5,000 cells per well), harvested in 10 days, and stained with crystal violet; the staining was extracted for the quantification of cell growth (right bottom). Data were represented as mean ± SD, * p < 0.001. B. Additional CRISPR vectors decrease the protein abundance of EGFR. Vectors encoding sg_EGFR_2 and sg_EGFR_3 were tested along with sg_EGFR_1 (sg_EGFR in Figure 6A). Protein lysates were harvested for immunoblotting. Cleaved PARP was used as a marker for apoptotic cell death, and Vinculin as a loading control. C. CRISPRing EGFR impairs cell proliferation. MDA-MB-468 cells were treated as in (A) for measurement of cell proliferation, * p < 0.001. D. Proliferation of TNBC cells (top, MDA-MB-468; bottom, DFBC12-58) treated with increasing concentrations of EGFR inhibitors or THZ1. Cells were harvested in three days for measurement of cell proliferation. E. TNBC cells (top, MDA-MB-468; bottom, DFBC12-58) were treated with vehicle control or indicated EGFR inhibitors for 30 min. Cell lysates were harvested for immunoblotting. See also Figure S6.