The roles of cyclin-dependent kinases (CDKs) in regulation of transcription and cell cycle Dalibor Blazek Transcriptional regulation group Molecular Medicine CEITEC-MU Summary • Kinases • Cyclin-dependent kinases (CDKs) • Cellular functions of cyclin-dependent kinase (CDKs)- in cell cycle - in transcription • Regulation of cyclin-dependent kinases (CDKs) • Role of cyclin-dependent kinases (CDKs) in human disease (cancer) • Cyclin-dependent kinase (CDKs) as a drug target in treatment of human disease (cancer) Kinases are enzymes • Kinases catalyze transfer of a phosphate from high energy molecule such as ATP to a substrate (protein) • Phosphatases are enzymes catalyze reversible reaction (dephosphorylation) Kinases are enzymes • 1954 first time observed phosphorylation of protein casein by Gene Kennedy • 1956 described reversible phosphorylation/dephosphorylation of proteins by Edmond Fischer and Edwin Krebs (Nobel prize 1992) Reversible phosphorylation regulates basic cellular processes and its deregulation leads to a disease Phosphorylation of proteins on four amino amino acids: serine (Ser) threonine (Thr) tyrosine (Tyr) histidine (His) Controls conformation and function of proteins Affects basic cellular processes: protein signaling pathways cellular metabolism cellular division cell cycle etc. (Deregulation of phosphorylation results in disease: cancer immune diseases etc.) Reversible phosphorylation regulates basic cellular processes and its deregulation leads to a disease Human kinases = group of Cyclin-dependent kinases (CDKs) Human kinome consists of 560 kinases divided into 8 groups Cyclin-dependent kinases (CDKs) Cyclin-dependent kinases (CDKs) Protein complexes that compose of: 1) CDK (kinase) subunit 2) Cyclin subunit Both subunits needed for the kinase activity of the complex Amino acid sequence preference motif for phosphorylation: S/T-P-X-K/R CDKs have at least one cyclin partner Sometimes cyclin subunit regulates specificity towards substrate, sometimes redundant In humans there are at least 20 genes encoding CDKs however only about half of the CDKs are sufficiently studied = relatively well studied Cdks Human cell has 20 CDKs and 29 Cyclins Regulation of cellular functions by CDKs The CDK complexes regulate various processes in cells Major functions: -Regulation of Cell Cycle (CDK1,2,4,6,7) -Regulation of Transcription (CDK7,8,9,11,12) Other functions: - regulation of pre-mRNA processing (CDK11, CDK9) - regulation of neuronal cell differentiation (CDK5) - likely more functions to be discovered CDK complexes regulate various processes in cells Regulation of Cell Cycle by CDKs Cell Cycle Cell cycle leads to production of two genetically identical daughter cells Major events of the cell cycle S-phase – DNA synthesis-duplication of the chromosomes M-phase – mitosis-pair of chromosomes segregated into the nuclei – cytokinesis- the cell divides into two identical cells The cell cycle has four phases G1 and G2 phases-time delay to allow the growth of the cell -time to monitor external and internal conditions before commitment to onset of S and M phase The control of the cell cycle: three major “checkpoints” Control of the cell cycle triggers essential processes such as DNA replication, mitosis and cytogenesis Cell cycle control system depends on cyclically activated CDKs that control three major “checkpoints” Cyclical changes (expression and degradation) in Cyclin protein levels result in cyclic assembly/disassembly and activation/inhibition of Cyc/CDK complexes; this leads to phosphorylation/dephosphorylation of proteins that initiate and regulate cell cycle events Cyclin protein levels change, CDK protein levels are constant Cyclinproteinlevels Cell cycle control system is a network of biochemical switches where Cyc/Cdk complexes play a major role Cyc/Cdk: Cell cycle phases: Event: Example: Mechanism of cell cycle arrest in G1 by DNA damage DNA damage causes transcription of p21, Cdk inhibitory protein, that inhibits G1-S- and S-Cdks, arresting the cell cycle in G1 phase Overview of cyclin/CDK-regulated cell cycle progression (2021) Overview of regulation of cell cycle machinery (cyclins and other proteins) by proteasomal degradation (2021) Evolution of cell cycle control Yeast- cell cycle is directed by one CDK: CDK1 (cdc28) Mammals-several CDKs (classical model), CDK1 is essential to drive cell cycle in the absence of other Cdk (mouse knock out model) Major Cyclins and CDKs in Vertebrates and Yeast Deregulation of cell cycle and cancer Cells escape from the proper control of the cell cycle during cancer development: -Increase in expression and activity of CDKs -Inactivation of inhibitors (regulators) of CDKs Consequences: Unscheduled proliferation Genomic instability Chromosomal instability Nobel prize (2001) for discovery of “key regulators of the cell cycle” Discovery of cyclin (early 1980s) Concept of “Checkpoint” (early 1970s) Discovery of CDK (mid 1970s) Regulation of transcription by CDKs Transcriptional Cyc/CDK complexes More CDKs in human versus in yeast likely reflects higher complexity of human genomes; i.e. human genes are longer, have introns etc more regulation needed Transcription (Gene expression) Transcription: synthesis of RNA from DNA template (gene) Transcription of protein-coding genes by RNA polymerase II (RNAPII) Promoter RNAPII RNAPII RNAPII Initiation Elongation Termination AAAA mRNA GenePromoter RNAPII Pre-initiating RNAPII CTD CTD CTD CTD C-terminal domain (CTD) of RNAPII plays a crucial role in regulation of transcription and co-transcriptional mRNA-processing Pre-mRNA CTD consists of 52 repeats of heptapeptide YSPTSPS in which individual amino acids get phosphorylated to form a “CTD code” (Y1-S2-P3-T4-S5-P6-S7)x52 P P P PP RNAPII CTD iso iso -52 repeats in humans (21 consensus, 31 non-consensus) -26 repeats in yeast -evolutionary conserved-important! Human “CTD code” RNAPII Roles of CDKs in the CTD phosphorylation (CTD code) (Y1-S2-P3-T4-S5-P6-S7)x52 Cdk9 Cdk7 P P P PP iso iso Cdk12 Cdk13 ? Cdk7Cdk9 ? Kinase X ? Modified CTD is a binding platform for transcription factors, RNA-processing factors and histone modification factors (code readers) RNAPII CTD Transcription factors RNA-processing factors Histone modification factors Exon Pre-mRNA Histones Phosphorylation of the CTD mediates: Transcription mRNA-processing Chromatin modifications RNA export Transcription-coupled genome stability CTD code “readers” For the regulation of transcription cycle the phosphorylations of the CTD by the Cyc/Cdks are essential Promoter RNAPII GTFs Cdk7 Promoter RNAPII GTFs RNAPII Cdk9 RNAPII RNAPII Pre-initiating RNAPII Initiated RNAPII Elongation Termination Ser2P AAAA Ser5P Mediator Capping enzyme Splicing/Chromatin remodeling Cleavage/PolyA factors factors Cdkx Cyclin/CDKs play a major role in regulation of transcription PresenceofCDKonagene Gene start Gene body Gene end DNA Cyclin/CDKs play a major role in regulation of transcription A parallel to cell cycle regulation; i.e. each stage of transcription is regulated by a different Cyclin/CDK pair In comparison to cell cycle CDK, the individual CDKs are NOT redundant Major differences between Transcription and Cell Cycle Cyc/CDK complexes Trancription Cyc/CDKs complexes: 1) Cyclin levels in cells do not oscilate (CDKs need to be constantly active for basal transcription) 2) Regulated at the level of recruitment to specific gene Ad 2) Examples of recruitment of P-TEFb (Cdk9) to genes P-TEFb=Cdk9 Deregulation of transcription and cancer Concept of “transcriptional addiction” of a cancer cell Regulation of CDKs kinase activity Kinase activity is crucial for many cellular processes Deregulation of kinase activity results in a disease The kinase activity is strictly regulated Regulation of kinase activity of CDK complexes Overview: Activation of CDK kinase activity: -Association of CDK with various Cyclin subunits -Phosphorylation of threonine in the “T-loop” of CDK -Degradation of CDK inhibitor proteins by ubiquitination and proteolysis Inhibition of CDK kinase activity: -Binding of CDK inhibitor proteins to Cyc/CDK complexes -Inhibitory phosphorylation of CDK -Ubiqitination and degradation of Cyclins in proteasome -Binding of CDK inhibitor proteins including small nuclear (sn)RNA to Cyc/CDK complex Activation of CDK kinase activity: -Association of CDK with various Cyclin subunits -Phosphorylation of Threonine in the “T-loop” of CDK T-loop blocks active site (active site=ATP binding site) T-loop moves out of the active site P-T-loop improves binding of substrate Activation of Cdk kinase activity-Cdk2-Cyclin A Activation of CDK kinase activity: -Degradation of CDK inhibitor proteins by ubiqitination and proteolysis Cell cycle-dependent phosphorylation of CDK inhibitor is a “mark” for recognition by SCF ubiquitin ligase, ubiquitinylation and degradation, rendering Cyc/CDK complex more active Inhibition of CDK kinase activity: -Binding of CDK inhibitor proteins to Cyc/CDK complexes P27 binding distorts and binds into the active site of CDK2 (for example inhibits G1/S-CDK in G1 phase) Inhibition of CDK kinase activity: -Inhibitory phosphorylation of CDK Inhibition of CDK kinase activity: -Ubiquitination and degradation of Cyclin by proteasome Mitosis-dependent activation of APC ubiquitin ligase leads to ubiquitination of Cyclin and its degradation Inhibition of CDK kinase activity: -Binding of Cdk inhibitor proteins and 7SK small nuclear RNA (7SK snRNA ) to CycT/CDK9 complex P-TEFb=CDK9 The kinase activity of CDK9 is inhibited by binding to several proteins and small nuclear RNA, 7SK snRNA Deregulation of transcription by CDKs leads to the onset of human diseases -HIV transcription- HIV specific Tat protein “steals” CDK9 from its cellular complex to transcribe HIV genome -Cancer - aberrant kinase activity of cell cycle and transcriptional CDKs (CDK4,6,7, 9,12) defective cell cycle and transcription HIV transcription is dependent on the CDK9 (P-TEFb) protein HIV Tat protein “steals” CDK9 from its complex with inhibitory Hexim1/7SK snRNA; resulting Tat/Cdk9 complex binds to HIV -TAR RNA element and drives HIV transcription in human cells HIV genome Integrated in human genome HIV Alterations of CDKs and their regulators in cancer CDKs and their regulators play a role as oncogenes and tumor suppressors Their alteration leads to a development of cancer Oncogene=gene that drives a cancer Tumor suppressor=gene that prevents a cancer Cell cycle CDKs/Cyclins are often amplified in various cancers (play a role of oncogenes)CDK4/CDK6 Cyclin D Cyclin E p21 RB1 Inhibitors/regulators of cell cycle CDKs/Cyclins are often deleted or mutated in various cancers (play a role of tumor suppressors) Transcriptional CDK12 can be either amplified or mutated in various cancers (oncogene / tumor suppressor) Transcriptional CDK12 is mutated in prostate and ovarian cancer KD=kinase domain The mutations lead to the aberrant kinase activity and defective transcription of certain genes important to preventing cancer (like DNA damage response genes-BRCA1, BRCA2 etc) CDK12 proposed to be a novel tumor suppressor W719del E928fs R882L E901C G909R K975E L996L T1014del Alterations in CDKs/Cyclins can be used as biomarkers for cancer treatment W719del E928fs R882L E901C G909R K975E L996L T1014del CDK12 mutations in ovarian cancer/breast: Indication for treatment with olaparib (PARP inhibitor) Inhibition of CDK activity is a attractive way to treat some diseases (cancer) • CDKs or cyclins are often amplified, overexpressed or activated in cancers (CDK4, CDK6, Cyclin E, Cyclin D) (CDK12, CDK9, CDK7, CDK8) Issues with kinase inhibitors • Selectivity-activity towards other kinases (ideally targeting only 1 kinase ) • Potency-concentration needed for inhibition of the kinase (IC 50 ideally in low nM) In practice: chemical inhibitors almost always inhibit other kinases Sometimes the outcome depends on inhibition of proper spectrum of kinases in a particular tumor Kinase domain structure and similarities within CDK family CDK2 Overlap of CDK2/CDK6/CDK7/CDK16 CDK inhibitors often have low selectivity within CDK family „quality kinase probe“ S. Knapp et al. Nat. Chem. Biol. 2013, 9, 3. • potent inhibition of primary target • at least 50 fold selectivity over other targets • demonstrated cellular activity data staurosporine imatinib (Gleevec) (FDA approved drug) only 22 (!) inhibitors passed Other protein kinase inhibitors: selectivity Kinase inhibition modes • ATP mimic (competitive) • Allosteric • Covalent • Degraders (PROTACs) Inhibitors: Kinase inhibition modes ATP mimic (competitive) inhibitors Kinase inhibition modes allosteric inhibitors Kinase inhibition modes Covalent inhibitors Kinase inhibition modes Degraders (PROTACs) Time-line of approved kinases inhibitors Food and drug administration (FDA) -approved kinase inhibitors mapped into the human kinome Timeline of the year for which agents with novel kinase family entered clinical trials Kinase targets of the small molecular inhibitors in clinical trials (2021) Indications, ongoing clinical trials and approved drugs (2021) Cost of bringing a single drug to the market Cost of bringing a single drug to the market -In average 2.6 billion USD (2019), 1 billion USD (2010) -Top 20 pharmaceutical companies spent 60 billion USD/year on drug development (2019) (Czech National budget was 66 billion USD in 2018) -97% of oncology drug candidates does not make it through clinical trials to drug approval mostly due to low efficacy and high toxicity (2019) Depletion of all 6 targets from cells did not stop the cancer growth! OTS964 works via inhibiting other kinase!!! (CDK11 is bona fide target of OTS964) Take home message: Importance of basic science to study molecular mechanisms of drug candidates OTS964 proliferation PBK proliferation PBKX Cyclin K/Cdk12: from identification of cellular function to synthesis of its first inhibitor/ set up of clinical trials Historically, Cdk9 and one of the cyclins (CycT1, CycT2 and CycK) were thought to form positive transcription elongation factor b (P-TEFb)-situation in 2008 Cdk9 Cyclin Cdk9 Cdk9 Cdk9 CyclinT1 CyclinT2 CyclinK P-TEFb Mostly studied Supports HIV transcription ? Not studied Does not support HIV transcription ? 2008 CycK binds Cdk12 2009 Cdk12 CycK CycK /Cdk12 is necessary for expression of DNA damage response genes 2011 Cdk12 was found among the most often somatically mutated genes in HGSOC Gene No. of Somatic Mutations (%) No. of Pubmed Papers Function P53 302 (96%) 63852 tumor suppressor BRCA1 11 (3%) 9231 tumor suppressor NF1 13 (4%) 3064 tumor suppressor CDK12 9 (3%) 27 ? BRCA2 10 (3%) 5793 tumor suppressor RB1 6 (2%) 2050 tumor suppressor 2011 Molecular characterization of CDK12 mutations in ovarian cancer 2015 Identification of the first inhibitor of CDK12 2016 Clinical trials evaluating CDK12 status as a biomarker In various cancer 2020 Clinical trials evaluating CDK12 status as a biomarker In various cancer-cont. 2020 Currently several biotech companies develop CDK12 inhibitors for clinical trials 2021