CDK6 Antagonizes p53-Induced Responses during Tumorigenesis

Abstract

Tumor formation is a multistep process during which cells acquire genetic and epigenetic changes until they reach a fully transformed state. We show that CDK6 contributes to tumor formation by regulating transcriptional responses in a stage-specific manner. In early stages, the CDK6 kinase induces a complex transcriptional program to block p53 in hematopoietic cells. Cells lacking CDK6 kinase function are required to mutate TP53 (encoding p53) to achieve a fully transformed immortalized state. CDK6 binds to the promoters of genes including the p53 antagonists Prmt5, Ppm1d, and Mdm4 The findings are relevant to human patients: Tumors with low levels of CDK6 have mutations in TP53 significantly more often than expected.Significance: CDK6 acts at the interface of p53 and RB by driving cell-cycle progression and antagonizing stress responses. While sensitizing cells to p53-induced cell death, specific inhibition of CDK6 kinase activity may provoke the outgrowth of p53-mutant clones from premalignant cells. Cancer Discov; 8(7); 884-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.

Figure 1. CDK6 supports the outgrowth of malignant cell clones

A) Cdk6^+/+ and Cdk6^-/- bone marrow cells were transduced with BCR-ABL. Numbers of apoptotic (red line) and living cells (black line) in liquid cultures are summarized in the left panels. The numbers represent the mean±SD (n=3 different biological replicates). B) Cell-cycle profiles of BCR-ABL-transduced bone marrow cells analyzed at day 22. Cdk6^+/+ cells display only a small sub-G1 fraction whereas Cdk6^-/- cells have a reduced number of cycling cells and an increased fraction of cell in sub-G1 phase. The gating strategy is indicated in the histogram plots; bargraphs summarize our experiments (n=3 different biological replicates). C) Statistics on the number of successfully established cell lines from BCR-ABL-transduced Cdk6^+/+ and Cdk6^-/- bone marrow cells (n=13 individual experiments). D) qPCR analysis of the p53-target genes p21, PUMA and NOXA in ex vivo γ-irradiated pre-pro-B cells isolated from Cdk6^+/+ and Cdk6^-/- mice at 4 hours post-treatment (n=3 different biological replicates). E) Western blot analysis of p53 and CDK6 in Cdk6^+/+ and Cdk6^-/- cell lines upon treatment with DMSO or etoposide for 4 hours. HSC-70 was used as loading control (n=3 different biological replicates). F) qPCR analysis of the p53-target genes p21, PUMA and NOXA in BCR-ABL⁺ Cdk6^+/+ and Cdk6^-/- cell lines upon treatment with NCS for 4 hours (n=3 different biological replicates). G) Kaplan–Meier plot showing overall survival of NSG mice after intravenous injection of Cdk6^+/+ (upper panel) or Cdk6^-/- (lower panel) BCR-ABL–transformed cell lines and subsequent γ-irradiation with 1.25 Gy or 2.5 Gy. Statistical differences were calculated using the Log-rank test (**p<0.01; ns: not significant; UT: Untreated). H) Drug screening of Cdk6^+/+ and Cdk6^-/- cell lines with 272 FDA approved drugs. Relative viability is indicated as % DMSO control (n=3 cell lines per genotype).

Figure 2. Cells lacking CDK6 kinase function are required to mutate p53

A) cDNA of the protein-coding p53 transcript was amplified by PCR and analyzed by Sanger-sequencing. The substituted amino acids are as well as the corresponding human mutations are indicated. B) Annexin V/7-AAD staining of Cdk6^-/- cells treated with etoposide, PRIMA-met or the combination of both drugs for 24h hours. Numbers represent the mean±SD (n=3 cell lines per genotype, one representative example is depicted). C) Cell-cycle profiles analyzed by PI-staining of BCR-ABL-transduced bone marrow cells at day 19. The gating strategy is indicated in the histogram plots; bargraphs summarize our experiments (n=3 different biological replicates). D) p53^+/+;Cdk6^+/+, p53^+/+;Cdk6^-/-, p53^-/-;Cdk6^+/+ and p53^-/-;Cdk6^-/- bone marrow cells were transduced with BCR-ABL. The panels show representative Annexin V/7-AAD stainings at day 19. The numbers represent the mean±SD (n=3 different biological replicates).

Figure 3. CDK6 induces a transcriptional program to antagonize p53

A) Heatmaps of transcripts deregulated in Cdk6^+/+, Cdk6^K43M and Cdk6^-/- colonies 10 days after BCR-ABL transduction (n=4 colonies per genotype). B) Gene ontology analysis of genes regulated in a kinase-dependent manner in colonies 10 days after BCR-ABL transduction (upper table). Examples of deregulated genes involved in the p53-reponse (lower table). C) qPCR analysis of the p53 regulators PRMT5 and MDM4 in individual colonies isolated from methylcellulose. D) Heatmaps of transcripts deregulated in BCR-ABL⁺ Cdk6^+/+, Cdk6^K43M and Cdk6^-/- cell lines (n=2 cell lines per genotype). E) Venn diagram showing the number of transcripts regulated in colonies and cell lines (blue circles) and the numbers of gene promoters with ChIP-Seq peaks (red circle). The numbers in the intersecting areas show the overlap between the two datasets and the genes having a ChIP-Seq peak in the promoter region. F) Representative examples of ChIP-Seq peaks in the promoter regions of the p53 antagonists Prmt5 and Mdm4. G) Pie charts showing the functional classification of gene ontology terms identified in the gene sets that are bound by CDK6 and show expression changes in colonies or cell lines.

Figure 4. Differential phosphorylation of transcriptional regulators upon CDK6 deficiency

A) Motif analysis of ChIP-Seq promoter peaks underlying CDK6-regulated genes. B) Schematic workflow of the technique used to detect phosphopeptides on chromatin (phospho-chromatome; LCMS: Liquid chromatography–mass spectrometry). C) Schematic representation of SP1 and NFYA proteins showing annotated domains and the position of residues that were found differentially phosphorylated in Cdk6^+/+ versus Cdk6^-/- cells. Transcriptional regulators showing reduced phosphorylation in the absence of CDK6 are listed (n=3 different biological replicates). D) Quantification of ADP formation in a luminescent kinase assay using recombinant NFYA and increasing amounts of active CDK6 protein. Photometrically acquired relative luminescence units (RFU) are shown (n=2 technical replicates). E) Luminescent kinase assay in the presence or absence of ATP or CDK4/6 inhibitor palbociclib (500nM) (n=2 technical replicates). F) NFYA chromatin immunoprecipitation (NFYA ChIP) followed by qPCR analysis of target gene promoters. Fold enrichment over the established Cd19 negative region is shown. G) ChIP of NFYA with subsequent Re-ChIP of CDK6. qPCR analysis shows the concomitant presence of CDK6 and NFYA at the indicated promoters. Fold enrichment over no-antibody control is shown.

Figure 5. CDK6 and p53 share common binding sites

A) Overlay of CDK6 with p53 ChIP-Seq peaks in untreated or γ-irradiated B-cells. The left panel depicts the distribution of peakshifts across CDK6 and p53 ChIP-Seq datasets. The Venn diagram depicts overlapping and distinct peak numbers in γ-irradiated B-cells. B) HA-CDK6 and p53 ChIP-qPCR experiments performed in BCR-ABL⁺ cell lines upon nutlin-3 pre-treatment (30µM, 4 hours). IgG-ChIP qPCR values were used as control. Upon nutlin-3 treatment, p53 shows increased binding to the Prmt5, Mdm4 and Ppm1d promoters paralleled by decreased CDK6-binding. C) Motif analysis of p53 ChIP-Seq peaks from γ-irradiated cells that overlap with CDK6 ChIP-Seq peaks (left) or are not found in the CDK6-ChIP-Seq dataset (right). D) Cumulative position-specific densities of motif found in p53 ChIP-Seq peaks from γ-irradiated cells that overlap with CDK6 ChIP-Seq peaks (left) or are not found in the CDK6-ChIP-Seq dataset (right). E) Schematic representation of the Prmt5, Ppm1d, Mdm4, Puma and Noxa promoter regions. The NFYA, SP1 and p53 binding sites and the identified motifs are indicated. F) Proposed model for the CDK6-dependent regulation of p53-responses via NFY/SP1 phosphorylation.

Figure 6. The CDK6-associated gene signature is conserved in human and murine leukemia

A) Gene set enrichment analysis of the p53-regulatory pathway in ALL and MDS patients. B) Heatmap of fRMA-normalized values from probes correlating with CDK6 expression in all of the analyzed leukemia-entities of the individual patients. To visualize co-expression with CDK6, patients within each datasets were sorted for increasing mean CDK6 levels. C) Scatter plots show the correlation between CDK6 expression and the expression levels of the indicated transcripts. Each dot indicates one patient. fRMA normalized expression values are shown. D) Percentages of ALL, AML and MDS patients with p53 mutations and mono-allelic loss of 7q. The chi-square test was used to test for statistical significance.