Transcription-Associated Cyclin-Dependent Kinases As Targets and Biomarkers for Cancer Therapy

Published OnlineFirst February 18, 2020; DOI: 10.1158/2159-8290.CD-19-0528

Review

Transcription-Associated Cyclin-Dependent Kinases as Targets and Biomarkers for Cancer Therapy

Jonathan Chou1,2, David A. Quigley1,3, Troy M. Robinson1,4, Felix Y. Feng1,2,4,5, and Alan Ashworth1,2

abstract Drugs targeting the cell cycle–regulatory cyclin-dependent kinase (CDK) 4 and 6 have been approved for the treatment of hormone receptor–positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Another class of CDKs, the transcription-associated CDKs, including CDK7, CDK8, CDK9, CDK12 and CDK13, are critical regulators of gene expression. Recent evidence suggests several novel functions of these CDKs, including regulation of epigenetic modifications, intronic polyadenylation, DNA-damage responses, and genomic stability. Here, we summarize our current understanding of the transcriptional CDKs, their utility as biomarkers, and their potential as therapeutic targets.

Significance: CDK inhibitors targeting CDK4 and CDK6 have been approved in hormone receptor–positive breast cancer, and inhibitors targeting other cell-cycle CDKs are currently in clinical trials. Several studies now point to potential therapeutic opportunities by inhibiting the transcription-associated CDKs as well as therapeutic vulnerabilities with PARP inhibitors and immunotherapy in tumors deficient in these CDKs.

INTRODUCTION subunit (RPB1) of RNA polymerase II (RNA Pol II), as well as other targets. However, their precise mechanisms of action The cyclin-dependent kinases (CDK) are a family of approx- related to transcription remain relatively obscure (1). In addi- imately 20 serine/threonine kinases that regulate fundamen- tion, there remains a class of CDKs for which the underlying tal cellular processes. They are broadly divided into two major functions are largely unknown. Each of the CDKs is bound to subclasses: (i) cell cycle–associated CDKs (including CDK1, a specific cyclin, which directs the activity of the CDK. Given CDK2, CDK4, and CDK6) that directly regulate progression that CDKs control processes critical for cancer cell survival through the phases of the cell cycle and (ii) transcription- and growth, they have been viewed as promising therapeutic associated CDKs (including CDK7, CDK8, CDK9, CDK12, targets. Indeed, multiple CDK inhibitors have been developed and CDK13). The transcription-associated CDKs regulate and tested in a number of cancer types (reviewed in ref. 1). gene transcription by phosphorylating the carboxy-termi- Recently, inhibitors that target CDK4/CDK6 (e.g., palboci- nal domain (CTD) of the DNA-directed RNA polymerase II clib, ribociclib, and abemaciclib) have become widely used in hormone receptor–positive [i.e., estrogen receptor (ER) and/

1Helen Diller Family Comprehensive Cancer Center, University of California, or progesterone receptor–expressing] breast cancer, and have San Francisco, San Francisco, California. 2Department of Medicine, Divi- shown impressive improvements in progression-free survival sion of Hematology/Oncology, University of California, San Francisco, (PFS) and overall survival (OS; refs. 2–4). Moreover, inhibitors San Francisco, California. 3Department of Epidemiology and Biostatistics, that target CDK1 and CDK2 are currently in clinical trials for 4 University of California, San Francisco, San Francisco, California. Depart- multiple cancer types (5). ment of Radiation Oncology, University of California, San Francisco, San Francisco, California. 5Department of Urology, University of California, In contrast, the transcription-associated CDKs are less San Francisco, San Francisco, California. developed as therapeutic targets, and small-molecule inhibi- Note: Supplementary data for this article are available at Cancer Discovery tors of transcription-associated CDKs have not yet entered Online (http://cancerdiscovery.aacrjournals.org/). routine clinical use. However, several recent studies implicate Corresponding Author: Alan Ashworth, UCSF Helen Diller Family Com- these CDKs in driving and maintaining cancer cell growth, prehensive Cancer Center, 1450 3rd Street, Box 0128, San Francisco, particularly in cancers primarily driven by dysregulated tran- CA 94158-0128. Phone: 415-476-5876; E-mail: [email protected] scription factors, such as those dependent on MYC (e.g., Cancer Discov 2020;10:1–20 neuroblastoma) or the EWS–FLI1 fusion oncoprotein (Ewing doi: 10.1158/2159-8290.CD-19-0528 sarcoma). Mounting evidence suggests that inhibiting this ©2020 American Association for Cancer Research. class of CDKs may have important therapeutic relevance.

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In addition, some transcription-associated CDKs, such as can also associate with the Mediator complex to regulate CDK12, are inactivated in ovarian and prostate cancers, sup- transcription in a gene-specific manner (25) and has kinase- porting a tumor-suppressive role for these transcriptional independent roles in regulating the p53 stress response (28). CDKs. Here, we review our current understanding of the A large-scale proteomics study recently identified more than transcription-associated CDKs and highlight the genomic 60 proteins phosphorylated by CDK8 and CDK19, many features of tumors carrying transcriptional CDK loss-of-func- of which are associated with chromatin modification, DNA tion mutations and potential synthetic lethal approaches. We repair, and transcription (29). discuss the utility of using transcription-associated CDKs CDK12 was discovered as a CDC2-related kinase with an as biomarkers for targeted therapies, potential combination arginine/serine rich (RS) domain (also known as CRKRS; ref. strategies with checkpoint immunotherapy, and the recent 30). The CRKRS/CDK12 gene encodes a protein of 1,490 amino development of small-molecule inhibitors against transcrip- acids and is one of the largest CDKs, encompassing a carboxy- tional CDKs in various cancer types. terminal kinase domain, two proline-rich motifs (PRM) involved in protein–protein interactions, and an RS domain that is commonly found in splicing factors of the serine/arginine-rich STRUCTURE AND FUNCTION OF family (ref. 30; Fig. 1A). Within the nucleus, CDK12 localizes in TRANSCRIPTION-ASSOCIATED CDKs a speckled pattern, overlapping with spliceosome components Each of the transcription-associated CDKs binds to an (30). CDK12 is essential during embryonic development, and activating cyclin partner to regulate gene transcription (6). knockout of the gene in mouse embryonic stem (ES) cells leads CDK7 (also known as CDKN7) is a 346 amino acid protein to lethality shortly after implantation; Cdk12−/− blastocysts fail (Fig. 1A) that binds to cyclin H and the accessory protein to undergo outgrowth of the inner cell mass due to apoptosis MAT1 to function as a CDK-activating kinase (CAK), and and exhibit increased spontaneous DNA damage (31). The has a general role in transcription: by phosphorylating search for cyclin partners initially identified cyclins L1 and L2 as the CTD of RNA Pol II, CDK7 regulates the initiation of cognate cyclins for CDK12 (32), but cyclin K was later shown to transcription and promoter escape (7, 8). The CDK7/CAK be the bona fide CDK12-associating cyclin critical for its kinase complex, which associates with the core TFIIH complex, activity (33–35). can activate CDK9 (PITALRE, CDC2L4, CTK1) by phos- The closest related CDK to CDK12 is CDK13 (also known phorylating the threonine-186 residue within the activating as CDC2L5, CHED), which is also a large CDK consisting T-loop. This cascade of events controls the switch from tran- of 1,512 amino acids. CDK12 and CDK13 share 50% amino scriptional initiation to elongation of RNA Pol II (9). Several acid identity overall, having unrelated amino and carboxy- recent reviews on have been published on the functions of terminal domains. CDK13 contains a carboxy-terminal ser- CDK7 (10, 11). ine-rich (SR) domain and two alanine-rich (AR) domains, CDK9, a 372 amino acid protein, binds to either cyclin T which are not found in CDK12 (ref. 36; Fig. 1A). However, or cyclin K for its kinase activity (12, 13). The CDK9 com- the kinase domains of CDK12 and CDK13 are nearly 92% plex with cyclin T, referred to as the positive transcription identical. elongation factor b (P-TEFb), is a general transcription fac- Structural studies of CDK12 and CDK13 have demon- tor (GTF) that is required for efficient expression of most strated that the amino-terminal lobe of the CDK12 kinase genes and phosphorylates the CTD of RNA Pol II, as well domain interfaces with the cyclin box of cyclin K, creating as the DRB sensitivity–inducing factor (DSIF) and negative CDK12:cyclin K heterodimers, which then further dimer- elongation factor (NELF), to relieve promoter pausing and ize (35). Similar heterodimers between CDK13 and cyclin K promote transcription elongation (14). CDK9 preferentially have also been observed (37). However, despite the similari- localizes to the nonnucleolar nucleoplasm with significant ties between CDK12 and CDK13, each seems to regulate the enrichment at nuclear speckles and is thought to also func- expression of a distinct set of genes (37, 38). These data sug- tion in RNA processing and replication stress responses (15, gest shared but nonoverlapping functions between CDK12 16). The structures of CDK7 and CDK9 have a typical kinase and CDK13. fold forming the amino terminal lobe (consisting of a β-sheet and α-helix), in addition to an α-helix–rich carboxy-terminal Other Transcriptional CDKs lobe (17). In addition to the CDKs that we have described above, CDK8, a 464 amino acid protein, associates with cyclin C, CDK10 and CDK11 also have presumed roles in transcrip- MED12, and MED13 to form a 600-kDa complex known as tion (39). CDK11 associates with splicing machinery as well the CDK8 module. CDK8 can associate with the Mediator as proteins involved in transcriptional initiation and elonga- complex, which is a multimeric transcriptional coactivator tion (40–42). Cyclin M is the cyclin partner for CDK10 (43), complex that transmits signals from transcription factors to and CDK11 associates with L-type cyclins (40). Whereas RNA Pol II (18–20). The CDK8/Cyclin C complex also targets CDK10 has been implicated as a tumor-suppressive kinase CDK7/cyclin H via phosphorylation, repressing the ability in estrogen-driven cancers (44), CDK11 is highly expressed in of TFIIH to activate transcription and its CTD kinase activ- triple-negative breast cancer (TNBC), multiple myeloma, and ity (21). CDK8 has also been described to restrain activation liposarcoma (45, 46). of superenhancers, affecting global gene expression in a cell type–specific fashion (22), and mediates responses to serum Roles in Transcription stimulation, inflammation, and hypoxia (23–26). CDK19 is The transcription-associated CDKs play major roles in the a paralog of CDK8 and also binds to cyclin C (27). CDK19 multistep process of RNA Pol II transcription. Functionally,

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CDK7 Kinase domain CDK12 RS PRM Kinase domain PRM

AA 12 295 346 AA 103 379 525 719 1051 1238 1490 697 1280

CDK8 Kinase domain CDK13 PRMARARS R Kinase domain PRM SR

34 62 189 434 449 697 1029 1220 1341 1512 AA 21 335 464 AA 57 76 489 1269 1403

CDK9 Kinase domain

AA 19 315 372

B Initiation Pausing/release Elongation Termination

Mediator complex

PIC CDK8 Cyc C RNA NELF RNA RNA RNA Pol II Pol II DSIF Pol II Pol II Ser2 phosphorylation GTFs mRNA mRNA Ser5 phosphorylation mRNA Ser7 phosphorylation Cyc K Cyc K CDK7 CDK9 CDK12 Cyc K Cyc H CDK12 MAT1 CDK13 BRD4 Cyc T1

C Ser2-P

CDK13

CDK12

CDK7 Ser7-P CDK9 ChIP enrichment

Ser5-P

TSS Gene body PolyA

Figure 1. Schematic structures of the transcription-associated CDKs and their roles in regulating RNA polymerase II. A, Schematic representation of the functional domains of the family of transcription-associated CDKs. CDK7, CDK8, and CDK9 consist primarily of the kinase domain only. In contrast, both CDK12 and CDK13 contain central kinase domains. CDK12 also has an amino-terminal RS and PRM domain, and a carboxy-terminus PRM domain. CDK13 contains an amino-terminal PRM and RS domain (order is flipped compared with CDK12) as well as two AR domains. The carboxy-terminal of CDK13 contains an additional PRM and SR domain. The functional significance of these domains is not well understood. The numbers denote the amino acid positions that form the boundaries of the protein domain. B, The transcription-associated CDKs regulate transcription by phosphorylating the CTD of RNA polymerase II (RNA Pol II) at serine 2, serine 5, and serine 7. In addition, CDK8 binds to the mediator complex to promote transcription activation and regulates gene expression from superenhancers. Blue dot, serine 2; red dot, serine 5; green dot, serine 7. C, Differential phosphorylation of the RNA Pol II CTD is observed at the transcriptional start site (TSS), along the gene body, and at the polyadenylation (polyA) site, as determined by chromatin immunoprecipitation (ChIP) studies. AA, amino acid; PRM, proline-rich motif; RS, arginine-serine rich domain; AR, alanine-rich motif; SR, serine-rich motif. Cyc C, Cyclin C; GTF, general transfection factor; PIC, preinitiation complex; NELF, negative elongation factor; DSIF, DRB sensitivity–inducing factor; BRD4, bromodomain-containing domain 4; Cyc T1, cyclin T1; Cyc K, cyclin K.

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REVIEW Chou et al. transcriptional CDKs and their cyclin partners control phos- 61). Specific sets of genes may be more sensitive to genetic phorylation of the RNA Pol II CTD (Fig. 1B and C). The CTD CDK12 inhibition, resulting in decreased Ser2 phosphoryla- is composed of multiple heptapeptide repeats (YSPTSPS) tion only at some gene loci (62), whereas chemical inhibition which can be phosphorylated (in humans, 52 repeats vs. of CDK12 and CDK13 appears to more globally dampen in yeast, 26 or 27 repeats; ref. 47). The length of the CTD Ser2 phosphorylation (60). In addition to the CTD, CDK12 partially determines the efficiency of processing different and CDK13 phosphorylate other substrates, which may be pre-mRNA substrates (47), whereas the pattern of tyrosine, critical for their functions in both transcription and other serine, and threonine phosphorylation dictates the transition processes; these targets have yet to be fully characterized but between the initiation, elongation, and termination phases of phosphoproteomics and affinity-purification mass spectrom- transcription (48). etry studies have identified multiple pre-mRNA processing Preceding transcription is the assembly of the preinitia- and RNA-splicing factors as potential kinase substrates and tion complex (PIC), a complex of roughly 100 proteins that protein-binding partners, although the exact consequences of docks to the transcription start sites of genes and facilitates phosphorylation remain unknown (38, 63). DNA entry into the active site of RNA Pol II for transcription Interestingly, CDK12 depletion does not affect transcrip- to start (49). Formation of the PIC requires the recruitment tion globally, but instead alters a select subset of genes of several GTFs, which include TFIIA, TFIIB, TFIID, TFIIE, involved in the DNA damage response (DDR) and DNA TFIIF, and TFIIH (6, 50). The final GTF to be recruited to replication (61, 64). Blazek and colleagues showed that the PIC is TFIIH, which contains multiple subunits, includ- loss of CDK12 decreases expression of predominantly long ing xeroderma pigmentosum type B (XPB) and CDK7 (51). genes (spanning >10 kb) with high numbers of exons, which Following recruitment, XPB functions as an ATP-dependent includes those involved in regulating genomic stability such DNA helicase that enables promoter opening for transcrip- as BRCA1, ATR, and FANCD2 (64). Loss of CDK12 results tion to occur (51, 52), whereas CDK7-mediated phospho- in reduced transcription of the BRCA1 gene and increases rylation of the RNA Pol II CTD at Ser5 may be involved in sensitivity to DNA-damaging agents (64). Moreover, muta- TFIIH-mediated promoter escape (11, 53). However, the pre- tions in the CDK12 kinase domain lead to an impaired cise role CDK7 plays in transcription remains controversial. ability to repair DNA double-strand breaks via homologous For example, it was recently shown that selective inhibition recombination (HR; refs. 64, 65). Recently, George and col- of CDK7 with YKL-5-124 does not significantly downregu- leagues demonstrated that inhibiting CDK12 and CDK13 late phosphorylation of the CTD or global gene expression, using a small-molecule inhibitor, THZ531, results in gene but instead primarily inhibits E2F-driven gene expression length–dependent elongation defects, and induces prema- and causes G1–S cell-cycle arrest (54). In addition, CDK7 ture cleavage and polyadenylation of DDR genes, leading may also indirectly regulate transcription by phosphorylat- to decreased DDR gene expression (63). CDK12 may also ing and regulating transcription factors (TF), including the regulate other CDKs, as disruption of Cdk12 in knockout ER and androgen receptor, two TFs that play critical roles in mice and P19 mouse teratocarcinoma cells reduces Cdk5 hormone-driven cancers such as breast and prostate cancers expression (66). Consistent with the notion that CDK12 (49–51). selectively regulates a subset of genes, studies in Drosophila Following initiation, RNA Pol II enters transcriptional cells showed that CDK12 is not required to maintain overall pausing, during which NELF and DSIF are loaded onto RNA transcription, but is required for the expression of NRF2- Pol II (48). Transcriptional pausing ensures gene-specific regu- dependent stress-response genes (67). Furthermore, gene lation, RNA quality control, and 5′ mRNA capping by the expression changes after knockdown of CDK13 or CDK12 human capping enzyme. The P-TEFb/CDK9 complex is then are markedly different, with enrichment of growth signaling recruited to paused RNA Pol II and cooperates with bromodo- pathways after CDK13 loss (37). In addition, although both main-containing protein 4 (BRD4) and the super elongation CDK12 and CDK13 directly interact with splicing machin- complex to release RNA Pol II for active transcription (Fig. 1B). ery, they affect the processing of different sets of genes and In this process, CDK9 phosphorylates the CTD, as well as DSIF noncoding RNAs (38). The basis for this specificity remains and NELF, to facilitate transcriptional elongation and recruit- poorly understood. ment of the 3′-end processing and splicing factors necessary for In addition to regulating the elongation phase of RNA mRNA maturation (55, 56). Several TFs (such as MYC) have Pol II, CDK12 also regulates transcriptional termination. been shown to recruit P-TEFb/CDK9 to promote transcription CDK12 deficiency reduces Ser2 phosphorylation of the CTD of their targets (57). CDK9 also binds to other transcriptional as well as levels of cleavage stimulation factor 64, a regula- mediators, such as the recombining binding protein suppressor tor of polyadenylation, at the 3′ end of the c-FOS gene; this of hairless (RBPJ) to target gene promoters, which maintains results in impaired 3′-end processing and expression (68). tumor-initiating cells in glioblastoma (58) and phosphorylates Taken together, these studies suggest that CDK12 plays other important targets, including the exoribonuclease XRN2, a role in transcription termination (Fig. 1B); impairing to regulate transcription termination (59). this function could result in the activation or loss of key In addition to CDK7 and CDK9, CDK12 and CDK13 also oncogenes or tumor suppressor genes, respectively, during phosphorylate the CTD at Ser2 and Ser5 in vitro and preferen- tumorigenesis. tially phosphorylate the CTD when it is prephosphorylated at Ser7 (refs. 30, 35, 37; Fig. 1B). However, there are conflicting Roles in Splicing data as to whether knockdown of CDK12 or CDK13 glob- Beyond regulating RNA Pol II phosphorylation, the ally decreases Ser2 and Ser5 CTD phosphorylation (38, 60, transcription-associated CDKs play other important roles

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Splicing Intronic polyadenylation Translational regulation

IPADPA mTORC1 DNA Raptor CHK1 mRNA tCDK IPA Cyc Cyc 3Ä tCDK tCDK 4E-BP1 Cap 5Ä Exon 1 Exon 2Exon 3Exon 4Exon 5 3Ä CDK12 intact CDK12 loss Release Loading Proximal ALE Distal ALE Translation factors

AAAAA AAAAA 4E-BP1

Cap Ribosome Exon 1Exon 2Exon 1Exon 2Exon 3Exon 4Exon 5

DNA replication and Epigenetic modification genomic stability Transcription-

H3K4me3 H3K36me3 associated CDK Mediator PIC A BCD complex RNA Pol II GTFs Tandem duplications

tCDK Cyc AABBCD

Figure 2. Beyond RNA Pol II phosphorylation: Transcription-associated CDKs regulate splicing, intronic polyadenylation, genomic stability, epigenetic modifications, and translation. In addition to their roles in phosphorylating the CTD of RNA Pol II, the transcription-associated CDKs have also been shown to: (i) control gene expression by interacting with splicing machinery to regulate alternative last-exon (ALE) splicing at proximal and distal ALEs; (ii) suppress intronic polyadenylation (IPA) usage, especially in long genes involved in homologous recombination repair, and maintain genomic stability; (iii) regulate epigenetic modifications and chromatin structure; and (iv) regulate translation. H3K4, histone-3 lysine-4; H3K36, histone-3 lysine-36; tCDK, transcription-associated CDK. Cyc, cyclin; DPA, distal polyadenylation.

(Fig. 2). Because CDK12 colocalizes with SC35 (also known the transcript followed by sequential addition of adenosine as SRSF2 or SFRS2; ref. 30), a component of the spliceo- residues, which is critical for nuclear export and stability some, it is thought to play a role in RNA splicing. CDK13 of the transcript, as well as for efficient translation into is also enriched in nuclear speckles, where the PRM domain protein (71). Interestingly, transcriptome sequencing has interacts with RNA-binding proteins within peri-nucleolar revealed that many genes can have more than one poly(A) structures (69). The smaller transcriptional CDKs (i.e., site; similarly to ALE splicing, differential polyadenylation CDK7, CDK8, and CDK9), which lack these additional thus allows cells to generate diverse transcript isoforms domains, likely do not participate in this function. Mul- with different 3′ ends, a process termed alternative poly- tiple mass spectrometry studies have shown that splic- adenylation (APA; ref. 71). A recent study in patients with ing machinery proteins are associated with CDK12 and chronic lymphocytic leukemia (CLL) uncovered widespread CDK13, including SRSF1 (38, 68–70). Support for a role upregulation of truncated mRNAs and proteins that were of CDK12 in splicing was further provided from studies generated by increased IPA, suggesting that this is an in breast cancer cell lines, where analysis of mRNA tran- epigenetic mechanism by which cancer cells can remodel scripts demonstrated that CDK12 regulates alternative last their transcriptomes and proteomes without altering their exon (ALE) splicing of a subset of genes, including those genomes (72). Truncated mRNAs were found predomi- involved in the DDR, to generate different mRNA isoforms nantly within tumor-suppressor genes, and the isoforms in their 3′ ends (70). Despite the evidence showing that generated by IPA usage lacked the normal tumor-sup- CDK12 and CDK13 are involved in splicing, the precise pressive functions found in the corresponding full-length mechanisms are not well understood. It remains unclear isoforms. Strikingly, some of the isoforms even gained how gene selectivity is achieved and what other protein de novo oncogenic potential, highlighting that global IPA partners are involved in this process. changes within a cell might be a newly recognized driver of tumorigenesis (72). Role in Suppressing Intronic Polyadenylation Dubbury and colleagues recently showed that CDK12 has Recent evidence suggests that in addition to a role in an important role in globally suppressing IPA usage to enable splicing, CDK12 may also be critical for regulating intronic production of full-length gene products (62). Using a genetic polyadenylation (IPA; Fig. 2). Polyadenylation is the process Cdk12 knockout model in mouse ES cells, the investiga- by which a poly(A) tail is added to the 3′ ends of eukaryotic tors demonstrated that Cdk12 loss results in the loss of HR mRNAs. This process involves endonucleolytic cleavage of repair genes and negatively affects transcription elongation

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REVIEW Chou et al. dynamics. Cdk12 loss also increased expression of p53 target several specific CDK12 “translation-only” target mRNAs genes, consistent with the induction of DNA damage. Inter- (76). These data suggest that in addition to the DDR and estingly, although ALE splicing was not dramatically altered stress response genes that require CDK12 for mRNA expres- genome-wide upon Cdk12 loss, isoform differences from APA sion and IPA suppression, another set of targets relies on usage were identified. In particular, proximal IPA sites were CDK12-mediated translational control, which may affect the significantly increased in HR genes (e.g.,Brca1 ), with a con- maintenance of genome stability. Whether CDK13 or other comitant decrease in distal polyadenylation sites. This was, transcription-associated CDKs also regulate translation is at least in part, due to the significantly more IPA sites in currently unclear. Nonetheless, taken together, it is becom- HR genes compared with other expressed genes. Interest- ing increasingly evident that the transcription-associated ingly, tumors harboring CDK12 loss-of-function mutations CDKs regulate gene expression not only by phosphorylating also had increased IPA sites in HR genes, suggesting that RNA Pol II, but also by modifying chromatin structure and the cumulative effect results in a functional HR-deficient controlling translation, thus expanding their functional phenotype, which may have therapeutic implications (see repertoires (Fig. 2). below). The increase in global intronic polyadenylation can also be recapitulated using THZ531 in neuroblastoma cells MOLECULAR ALTERATIONS IN and preferentially affects long genes involved in DDR (63). TRANSCRIPTION-ASSOCIATED Therefore, CDK12 has a further mechanism of regulating CDKs FOUND IN CANCER gene expression, namely by suppressing IPA and promoting distal (full-length) isoform expression, particularly in long Summary of Mutations, HR genes (Fig. 2). Amplifications, and Deletions Genomic alterations affecting CDK7, CDK8, CDK9, or Role in Chromatin, Epigenetic, and CDK13 are relatively uncommon in human cancers. The Translational Regulation association of these kinases to tumor development has gen- Dynamic regulation of chromatin structure can signifi- erally been through large-scale genetic screening approaches cantly alter gene expression. Two basic forms of chroma- or hypothesis-driven investigation, rather than direct obser- tin exist in eukaryotes: Euchromatin regions are generally vation of recurrent somatic events in human cancers (77–79). associated with open chromatin configurations and contain However, copy-number changes as manifested by gene ampli- transcriptionally active genes, whereas heterochromatin fications as well as deletions in these transcription-associated regions are typically less accessible to the transcriptional CDKs can be found, albeit rarely in human cancers (Fig. 3A). machinery due to the highly compacted structure. Loss of For example, the genomic locus harboring CDK8 is gained Cdk12 in Drosophila results in the ectopic accumulation of or amplified in roughly 20% of colorectal adenocarcinomas, HP1 on euchromatin regions, which leads to downregula- where CDK8 expression has been linked to β-catenin signal- tion of target genes (73). Interestingly, the heterochromatin ing (78). CDK12 has also been found to be coamplified with enrichment mainly occurs within long genes, and results in the ERBB2/HER2 oncogene in subsets of breast cancer (80). decreased transcription of neuronal genes involved in court- Interestingly, deletions and mutations in the cyclin-binding ship learning (73). This suggests that CDK12 regulates the partners for these CDKs have also been identified in human conversion of euchromatin to heterochromatin, highlight- cancers. For example, mutations in cyclin C (encoded by ing another distinct mechanism that CDK12 regulates in the CCNC gene) are found in a subset of acute lympho- gene expression. Future work investigating whether this blastic leukemias (ALL; refs. 81, 82). Cyclin C, which binds also occurs in human cancer cells will be important. Fur- to CDK8 as part of the Mediator complex, functions as a thermore, CDK7 has also been shown to regulate transcrip- haploinsufficient tumor suppressor by controlling Notch1 tion-associated chromatin modifications. CDK7 stimulates levels, and loss of CCNC accelerates tumor development in the methyltransferase activity of SETD1A/B to regulate a mouse model of T-cell ALL (T-ALL; ref. 82). The relative H3K4me3 downstream of the transcriptional start site, and scarcity of recurrent somatic alterations, either activating may indirectly regulate H3K36me3 positioning through or inactivating, observed in these kinases may be related to its effects on CDK9 and SETD2 (74, 75). Through these their dramatic influence on transcriptional activity of large mechanisms, CDK7 appears to dually regulate transcrip- numbers of downstream targets, which may both positively tional processing and gene methylation, suggesting that and negatively regulate cell growth, depending on the cellu- dysregulation of CDK7 might lead to heritable changes in lar context. However, CDK12 is exceptional in this regard: Of gene expression. the transcription-associated CDKs, it is the only one where Beyond regulating transcription and epigenetics, some inactivating somatic alterations (i.e., frameshift, nonsense, of these transcription-associated CDKs may play additional and missense mutations) have been recurrently observed roles in regulating protein expression at the translational in prostate and high-grade serous ovarian carcinomas (Fig. level. Choi and colleagues recently described a role for 3B and C) and where gene inactivation is linked to unique CDK12 in controlling translation of a subset of mRNAs genomic alterations. involved in DNA repair, translation regulation, and mitosis (76). For example, CDK12 phosphorylates the translation Genomic Characterization Reveals a Unique repressor 4E-BP1, which releases it from eIF4E at the 5′ Signature in CDK12-Mutant Tumors cap of mRNAs, enabling expression of mTOR complex 1 Numerous studies have identified mutations in theCDK12 (mTORC1) targets. Genome-wide ribosome profiling identified gene in human cancers. For example, in The Cancer Genome

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A 3 2 CDK7 1 0 −1 3 2 CDK8 1 0 −1 3 2 CDK9 1 0

copy number −1 2 3 CDK12

Log 2 1 0 −1 3 CDK13 2 1 0 −1 ov lgg lihc kirc acc ucs kirp tgct kich lusc laml blca chol dlbc sarc uvm luad stad thca brca gbm prad cesc esca hnsc ucec thym pcpg paad skcm meso coadread

Missense R858W CDK12 12% Mutation Amplification 5 D918G Deep deletion Multiple alterations G677Y 10% G677D R1008W G677C R773C R1008Q (2x) 8%

6% 0 RS PRM Kinase PRM 4%

2% Number of observations Frameshift/stopgain CDK12 alteration frequency

5 Breast OvariancancerProstate cancer adenocarcinom 0 200 400 600 800 1,000 1,200 1,400 Residue

Figure 3. Frequency and characteristics of transcriptional CDK alterations in human cancer. A, Copy number estimates of CDK7, CDK8, CDK9, CDK12, and CDK13 in human cancer. Plots depict log2 copy number estimates of CDK7, CDK8, CDK9, CDK12, and CDK13 across 10,702 tumors in 32 types of tumors, derived from data generated by the TCGA Research Network (https://www.cancer.gov/tcga). acc, adrenocortical carcinoma; blca, bladder cancer; brca, breast cancer; cesc, cervical squamous cell carcinoma and endocervical adenocarcinoma; chol, cholangiocarcinoma; coadread, colorectal adenocarcinoma; dlbc, diffuse large B-cell lymphoma; gbm, glioblastoma; hnsc, head and neck squamous cell carcinoma; kich, kidney chromophobe; kirc, kidney renal clear cell carcinoma; kirp, kidney renal papillary cell carcinoma; laml, acute myeloid leukemia; lgg, brain lower grade glioma; lihc, liver hepatocellular carcinoma; luad, lung adenocarinoma; lusc, lung squamous cell carcinoma; meso, malignant pleural mesothelioma; ov, ovarian serous cystadenocarcinoma; paad, pancreatic adenocarcinoma; pcpg, pheochromocytoma and paraganglioma; prad, prostate adenocarcinoma; sarc, Ewing sarcoma; skcm, melanoma; stad, stomach adenocarcinoma; tgct, testicular germ cell cancer; thca, papillary thyroid carcinoma; thym, thymic epithelial tumors; ucec, uterine corpus endometrial carcinoma; ucs, uterine carcinosarcoma; uvm, uveal melanoma. B, Records in cBioPortal (containing 41,666 unique patient records with mutation data; refs. 157, 158) were aggregated with high-grade serous ovarian carcinoma cases reported in Popova and colleagues (93) and metastatic prostate cancer cases reported in Wu and colleagues (98). The location and nature of these somatic alterations (indel, stop-gain, missense, splice, or translocation) were tabulated. Residue R858, the most frequently mutated amino acid in CDK12, is one of two canonical CDK arginines (35) that mediates stabilization of the T-loop to allow full activation of the kinase (65). In addition, residue R773 is important in forming electrostatic contacts with its cyclin partner, cyclin K. PRM, proline-rich motif; RS, arginine-serine rich domain. C, Frequency and types of somatic CDK12 gene alterations in breast, ovarian, and prostate cancers. Green, mutations; red, amplifications; blue, deep deletions; gray, multiple alterations.

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Atlas (TCGA) analysis of 489 high-grade ovarian adeno- ovarian carcinoma (modes of 400 Kb and 2.4 Mb). In contrast carcinomas, CDK12 was found to be one of 9 recurrently to HGSOC, where a majority of CDK12-inactivated tumors mutated genes, occurring in 3% of primary ovarian tumors were tetraploid, and single mutations with loss of heterozy- (83). BRCA1, BRCA2, and CDK12 mutations are mutually gosity was observed, a typical CDK12-inactivated mCRPC exclusive (84–86), but despite the mounting evidence that genome was diploid and frequently harbored two distinct CDK12 regulates the expression of HR genes, the genomic inactivating CDK12 somatic alterations. CDK12 inactiva- signature of CDK12-mutated tumors is unique from those tion was mutually exclusive with other established mCRPC with BRCA1 or BRCA2 mutations (87, 88). This suggests that genomic subclasses such as BRCA2 mutation, activating ETS- although CDK12 loss may share overlapping features with family gene fusions, or mutations in SPOP (98). This study BRCA1 or BRCA2 loss, our understanding is likely incom- was complemented by whole-genome studies of mCRPC that plete. Elucidating the roles of CDK12 in DNA replication noted that whereas BRCA2 inactivation produced both a may be important in understanding the genomic signatures characteristic structural signature of frequent deletions and seen in CDK12-mutated tumors. a nucleotide mutation signature, biallelic CDK12 inactivation was associated only with a structural TDP signature (87, 88). Focal Tandem Duplications in The mechanism by which these CDK12-associated TDs are CDK12-Mutated Cancers generated remains largely unknown. The cell-cycle CDKs in Genomic studies have uncovered tumors with a distinctive general prevent reinitiation of replication origins through mul- tandem duplicator phenotype (TDP), whereby tumors harbor tiple overlapping mechanisms, so that the genome is replicated hundreds of small copy-number gains, without an obvious only once in each cell cycle (99). Deregulation of the replica- selection for a single coding or noncoding location (89–92). tion initiation proteins MCM2-7 and CDC6 and induction of Tandem duplications (TD) are a structural rearrangement that rereplication aberrantly in yeast are sufficient to start the ini- produces physically contiguous, head-to-tail duplications of tial steps of TD formation (100). Rereplication-induced gene a segment of DNA. In contrast to tumors that develop focal amplifications appear to be mediated by nonallelic homolo- DNA copy-number gains targeting driver genes such as ERBB2/ gous recombination between repetitive elements. Interestingly, HER2, the apparent lack of selective pressure targeting a single cyclin K and CDK12 promote prereplication complex (pre-RC) locus in tumors with the TDP suggests a systemic etiology assembly in G1 by phosphorylating and restricting cyclin E1 rooted in a defect in DNA repair or replication. Interestingly, activity, and knockdown of cyclin K or CDK12 prevents assem- tumors with the TDP lack germline or somatic inactivation of bly of the pre-RC (101). One possibility is that loss of CDK12 BRCA2, indicating another unknown defect was responsible. causes dysregulation of replication origins, leading to TDs Using retrospective analysis of copy-number data from that accumulate over cell divisions. Interestingly, CDK12 was high-grade serous ovarian carcinomas (HGSOC), Ng and recently shown to be critical for proper chromosome align- colleagues showed that approximately 12% of HGSOC was ment and progression through mitosis by regulating mitotic characterized by the TDP phenotype (90). CDK12 inactiva- regulators such as the structural maintenance of chromosome tion was associated with 200 to 800 DNA copy-number gains complexes, centromere proteins (CENP), and the kinetochore per tumor, and the distribution of the TD sizes was bimodal, protein NDC80 (76). Furthermore, BRCA1 (but not BRCA2) with modes of 300 kb and 3 Mb (93). Some cases showed an was recently shown to suppress TDs, and microhomology- acquired somatic mutation in CDK12 and loss of heterozy- mediated TDs of approximately 10 kb are commonly found in gosity of the remaining functional allele (93). Subsequently, BRCA1-mutated cancers (102). Willis and colleagues showed Menghi and colleagues reanalyzed 992 TCGA genomes and that in the setting of BRCA1 loss, TDs might arise by a replica- formally reidentified the TDP by statistical analysis, report- tion restart–bypass mechanism terminated by end joining or ing TDs in three recurrent, narrowly defined duplication by microhomology-mediated template switching, the latter spans present in 12% of TCGA tumors (94). The TDP occurs leading to the formation of complex TD breakpoints (102). in up to 50% of triple-negative breast adenocarcinoma, ovar- There are conflicting reports on whether the TDP prefer- ian carcinoma, and endometrial carcinoma; 10% to 30% of entially targets specific loci for gene amplification or consist- adrenocortical, esophageal, stomach, and lung squamous ently disrupts tumor suppressors. The balance of evidence cell carcinoma, and less frequently in other tumor types (94). suggests that although the TDP affects the entire genome at Interestingly, in localized prostate cancer, both alleles of random, variants in cells that inactivate tumor suppressors CDK12 are inactivated in approximately 1% to 2% of cases or amplify relevant drivers will be selected and expanded. (89, 95). However, more than 5% of advanced prostate adeno- Menghi and colleagues demonstrated that the smaller TDs carcinomas harbor biallelic CDK12-inactivating mutations, associated with functional inactivation of BRCA1 and TP53 emphasizing that advanced prostate cancer is a genomically were more likely to disrupt genes by double transection distinct entity from primary disease (96). Loss of CDK12 within the coding region, whereas longer duplications such as in prostate cancer is also associated with features of more those associated with CDK12 were more likely to amplify cod- aggressive disease, including higher Gleason score and shorter ing and noncoding gene-regulatory elements (103). Wu and time to developing castration resistance and metastasis (97). colleagues observed TD-mediated amplification of the driver Focusing on CDK12 in metastatic castration-resistant pros- genes MCM7, RAD8A, CDK18, and CCND1 more frequently tate cancer (mCRPC), Wu and colleagues reported biallelic than expected by chance, with a dose-dependent increase in inactivation of CDK12 in 7% of mCRPC cases (98). CDK12 CCND1 expression (98). Gene rearrangement may also be an inactivation in this setting was also associated with a TDP, important consequence of the TDP, as the TDP in mCRPC with a bimodal size distribution similar to that reported in was associated with an increase in TD-associated fusion

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Transcription-Associated CDKs in Cancer REVIEW events, whereas in CDK12-intact tumors, fusions typically in approximately 10% of cases. As might be expected, CDK12 derive from translocations or large intragenic deletions (98). expression correlated with HER2 expression and the absence of CDK12 expression correlated with the TNBC phenotype TRANSCRIPTION-ASSOCIATED CDKs and reduced expression of a number of DNA-repair pro- AS BIOMARKERS AND POTENTIAL teins (80). Proteomic analysis of breast cancers demonstrated THERAPEUTIC VULNERABILITIES that amplification ofCDK12 is associated with enhanced CDK12 phosphorylation, suggesting that CDK12 may be CDK12 Loss as a Biomarker for Platinum, PARP an important therapeutic target in HER2-amplified breast Inhibitors, and Other Targeted Agents cancers (109, 110). However, functional validation in tumor Synthetic lethality has been utilized as a strategy to target cell lines overexpressing CDK12 is required to demonstrate cancers with germline or somatic loss-of-function mutations dependency on CDK12. Complicating matters, in some cases, in BRCA1 or BRCA2. Indeed, PARP inhibitors (PARPi) are the CDK12 gene may become rearranged within the HER2 FDA-approved drugs that target tumors with defects in HR, amplicon, potentially leading to the loss of CDK12 function including those with BRCA1 or BRCA2 mutations (104, 105). (111). In addition, a CDK12 gene fusion has been described in Given the success of this approach to target molecular sub- a rare micropapillary form of breast cancer (111). Therefore, sets of cancers, this therapeutic strategy has been extended whether CDK12 gene amplification is an oncogenic driver (or beyond BRCA and PARP, with the hope of targeting addi- simply a passenger) and whether this genetic alteration has tional tumors characterized by the loss of tumor-suppressor any therapeutic significance remains unclear at the moment. genes. However, with the exception of CDK12, deleterious loss-of-function mutations in the other transcription-associ- Ongoing Clinical Trials for ated CDKs have not been described in cancer. CDK12-Mutated Cancers A number of studies have now suggested that CDK12 muta- There has been recent interest in using CDK12 mutational tion or deficiency may lead to sensitivity to PARPi and platinum status as a biomarker for PARPi response (Table 1). TRITON2 chemotherapy as well as agents that target cell-cycle check- (NCT02952534) is a phase II study of rucaparib in patients points such as CHK1 (refs. 84, 106, 107; Fig. 4). For example, with mCRPC with HR gene alterations. The preliminary results a high-throughput genome-wide short-hairpin RNA (shRNA) were recently reported, which included 13 patients with CDK12 screen previously identifiedCDK12 as one of the most sig- alterations, in addition to 45 patients with BRCA1/BRCA2 nificant genes enhancing sensitivity to PARPi when depleted alterations and 18 patients with ATM alterations (112). In the (84). Given the role of CDK12 in the maintenance of genomic patients with CDK12 mutations, only 1 of 13 patients had a stability, it seems likely that PARPi target CDK12-mutant confirmed prostate-specific antigen (PSA) response (defined as tumors by further interfering with DNA repair. However, a sustained 50% decrease in baseline PSA), and no patients had the possibility that PARPi block the transcriptional function an objective response as defined by RECIST. The exactCDK12 of PARP1, which may be critical in the absence of CDK12, mutations were not reported, and will be critical in determin- may also be important. Indeed, PARP1 was previously shown ing how mutations in different domains of CDK12 may affect to regulate androgen receptor transcriptional activity and sensitivity to PARPi. function (108). In addition, data from TCGA showed that In addition to TRITON2, another trial, BRCAAway, is test- patients with CDK12 mutations have better responses to ing abiraterone (an inhibitor of androgen synthesis) versus platinum-based chemotherapy (84). Joshi and colleagues olaparib (a PARPi) versus the combination of abiraterone also demonstrated increased sensitivity to platinum and the plus olaparib in patients with mCRPC. This trial will include PARPi veliparib in ovarian cancer cell lines in which CDK12 a cohort of patients with CDK12 mutations (NCT03012321). was silenced. Several CDK12 point mutations within the A trial testing the combination of olaparib and copanlisib kinase domain disable the kinase activity of CDK12, suggest- (a PI3K inhibitor) plus an anti–PD-1/PD-L1 checkpoint ing that this domain is important for its tumor-suppressive inhibitor in molecularly selected patients will soon open function (106). (NCT03842228). One cohort will also include patients har- CDK12-deficient cells also appear to depend on the S-phase boring CDK12 mutations. Finally, a phase Ib trial is testing checkpoint kinase CHK1, and CHK1 inhibitors can selectively the combination of rucaparib plus irinotecan in patients with kill CDK12-deficient cells regardless ofTP53 status (107). The DNA-repair mutations, including CDK12 (NCT03318445; mechanism may involve CDK12 regulation of CHK1 itself; Table 1). It remains to be determined whether the laboratory CDK12-mutant ovarian cancers appear to have reduced CHK1 data in cell lines showing PARPi sensitivity in the setting of expression, which may underlie their sensitivity to CHK1 CDK12 loss will yield meaningful clinical advances in CDK12- inhibition. mutated cancers. In breast cancer, ERRB2/HER2 is an oncogene that is frequently amplified and overexpressed, and can be therapeuti- CDK12 Loss as a Biomarker for Immunotherapy cally targeted with the antibody trastuzumab. Because the Given the focal TDs and genomic instability seen within CDK12 gene is located close to the HER2 gene at chromosome CDK12-mutated prostate cancer genomes, it was hypothe- 17q12-q21, it is often coamplified withERRB2/HER2 and sized that these TDs might generate neofusion peptides that therefore may also be overexpressed. Naidoo and colleagues could stimulate and be recognized by the immune system. assessed the frequency of CDK12 overexpression by IHC in Wu and colleagues demonstrated that the genomic signature breast cancer. Twenty-one percent of primary unselected of CDK12 deficiency in advanced prostate cancers was differ- breast cancers were CDK12-high, and the protein was absent ent from that generated by either BRCA2 loss or mismatch

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CDK12 mutation

Deregulated RNA Pol II phosphorylation, Genomic TDs decreased DDR gene expression

DDR gene regulation

RNA Promoter Pol II Neoantigen fusions

5′ DDR genes Immune cell Cancer cell Cyc tCDK Neoantigens

TDs

Impaired DNA damage repair signaling

Checkpoint immunotherapy

PARP1 or CHK1 inhibitor

Figure 4. Therapeutic vulnerabilities in CDK12-mutated tumors. Given its role of regulating RNA Pol II and DDR gene expression, CDK12 deficiency or mutation has been shown to result in synthetic lethality with PARP inhibitors and CHK1 inhibitors in vitro. Targeting CDK12-mutated tumors with PARP inhibitors is currently being tested in multiple clinical trials (see Table 1). In addition, CDK12-mutated cancers harbor recurrent TDs, which have been postulated to lead to gene fusions. These TD gene fusions may function as neoantigens and increase the sensitivity to checkpoint immunotherapy (e.g., anti-CTLA4 and anti–PD-1/PD-L1 checkpoint inhibitors).

repair deficiency (MMRD; ref. 98). They showed that TDs of 4 patients with mCRPC, 50% of patients with CDK12 in coding regions generated a large number of gene fusions mutations responded to an anti–PD-1 checkpoint inhibitor. that could potentially function as neoantigens. Although These data suggest that the genomic instability resulting prostate cancer is generally thought of as a immunologically from CDK12 deficiency leads to an increase in neoantigens, “cold” tumor type (with the exception of prostate cancers and that this subtype of cancer may benefit from immune- harboring MMRD and microsatellite instability), prostate checkpoint inhibition (ref. 113; Fig. 4). This hypothesis is cancers harboring CDK12 mutations had a significantly being tested in multiple clinical trials, including a phase II increased number of T-cell infiltrates and expanded T-cell study of ipilimumab (targeting the CTLA4 checkpoint) plus clonotypes, similar to MMRD prostate cancers (98). Con- nivolumab (targeting the PD-1 checkpoint) in patients with sistent with the hypothesis that CDK12-deficient prostate CDK12-mutated metastatic cancers (NCT03570619; Table cancer might be more immunogenic, in a preliminary cohort 1). Moreover, whether the combination of immunotherapy

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Table 1. Clinical trials evaluating CDK12 mutational status as a biomarker in various cancer types

Therapeutic Trial name CDK biomarker intervention Objectives BRCAAway: A Randomized phase II trial of Patients with mCRPC and muta- Olaparib, Evaluate the objective PFS of abi- abiraterone, olaparib, or abiraterone + tions in noncanonical DNA repair abiraterone raterone/prednisone, olaparib olaparib in patients with metastatic cas- genes including CDK12 or the combination abirater- tration-resistant prostate cancer with one/prednisone + olaparib in DNA-repair defects (NCT03012321) patients with mCRPC TRITON2: A multicenter, open-label Patients with mCRPC and muta- Rucaparib Evaluate the ORR and PSA re- phase II study of rucaparib in patients tions in noncanonical DNA repair sponse in patients with mCRPC with metastatic castration-resistant genes including CDK12 prostate cancer associated with homologous recombination deficiency (NCT02952534) IMPACT: Immunotherapy in patients with Patients with mCRPC or other can- Ipilimumab plus Evaluate the ORR and PSA re- metastatic cancers and CDK12 muta- cers and CDK12 loss-of-function nivolumab sponse in patients with mCRPC tions (NCT03570619) mutations Nivolumab in biochemically recurrent Patients with biochemically recur- Nivolumab Evaluate the PSA50 response as dMMR prostate cancer (NCT04019964) rent prostate cancer after prior well as the PSA PFS, metas- local therapy and no radiographic tasis-free survival, and time evidence of metastasis and to initiation of next systemic CDK12-inactivating mutations therapy or dMMR Phase II trial of PARP inhibitor niraparib Patients with high-risk localized Niraparib Evaluate the tumor stage, lymph for men with high-risk prostate cancer prostate cancer and mutations in node metastasis, margins, and and DNA damage response defects canonical and noncanonical DNA pathologic CR rate at prosta- (NCT04030559) repair genes including CDK12 tectomy and PSA PFS Combination therapy of rucaparib and Patients with advanced cancer Rucaparib plus Evaluate the ORR as defined by irinotecan in cancers with mutations in and mutations in canonical and irinotecan the proportion of patients with DNA repair (NCT03318445) noncanonical DNA repair genes either confirmed CR or partial including CDK12 response (as per RECIST) ORCHID: Phase II study of olaparib in Patients with renal cell carcinoma Olaparib Evaluate the ORR as defined by patients with metastatic renal cell car- and mutations in DNA repair the proportion of patients with cinoma harboring a BAP1 or other DNA genes including CDK12 either confirmed CR or partial repair gene mutations (NCT03786796) response (as per RECIST) A phase Ib biomarker-driven combina- Patients with advanced metastatic Copanlisib, Evaluate the MTD of copanlisib tion trial of copanlisib, olaparib, and cancer and germline or somatic olaparib, plus and olaparib. Secondary MEDI4736 (durvalumab) in patients with mutations in DNA damage repair durvalumab objectives include assessment advanced solid tumors (NCT03842228) genes, including CDK12 of the ORR as defined by the proportion of patients with either confirmed CR or partial response (as per RECIST)

Abbreviations: CR, complete response; dMMR, deficient in mismatch repair; ORR, overall response rate.

with a PARPi might be efficacious inCDK12- deficient cancers transcription for oncogenesis (i.e., “transcriptionally addicted” awaits further investigation. cancers) has fueled interest in directly targeting these CDKs. Moreover, increased global transcription is a putative mechanism for oncogene-induced DNA damage, suggesting that high CHEMICAL INHIBITORS OF levels of transcription may be linked to genomic stability (114). TRANSCRIPTION-ASSOCIATED CDKs Given the critical roles that transcription-associated CDKs CDK7 Inhibitors in Transcriptionally play in regulating gene expression, they have emerged as puta- Addicted Cancers tive targets in cancer and other diseases (reviewed in ref. 57). The Gray and colleagues initially discovered and characterized recognition that certain tumors are dependent on persistent a covalent CDK7 inhibitor called THZ1, which targeted a

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REVIEW Chou et al. remote cysteine residue located outside the canonical kinase mediator of hypoxia-inducible factor 1 (HIF1A) target gene domain, providing some selectivity for CDK7, although expression (23). In addition, CDK8 promotes transcription THZ1 also has weak activity against CDK9, CDK12, and of genes involved in glycolysis, and inhibition of CDK8 CDK13 (115). Recently, Gray and colleagues described a reduces glucose uptake and sensitizes cancer cells to glyco- more potent and selective covalent CDK7 inhibitor, YKL- lysis inhibitors (119). 5-124. Interestingly, although treatment with YKL-5-124 In colon cancer, CDK8 is located in a region of the genome led to a G1–S cell-cycle arrest, there was little effect on RNA that shows recurrent copy-number gains, and is an onco- Pol II phosphorylation status, suggesting some functional gene that acts in part by regulating β-catenin activity and redundancy among the transcriptional CDKs (54). A subset WNT signaling (78). CDK8 expression is associated with of cancer cell lines, including human T-ALL, demonstrated poor clinical outcomes in colon cancer (120). In addition, remarkable sensitivity to CDK7 inhibition, in part by affect- CDK8 protects β-catenin–dependent transcription from inhi- ing RUNX1 transcription (115). The potential to disrupt bition by the E2F1 transcription factor, which antagonizes transcriptionally addicted cancers via CDK7 inhibition was the WNT pathway (121). However, conflicting data exist on later supported by studies showing that MYCN-amplified whether CDK8 is essential for survival, as genetic ablation neuroblastoma cells and MYC/MYCL–amplified small-cell has little effect on proliferation in cell-line models of colon lung cancer (SCLC) cells were also sensitive to CDK7 inhibi- cancer, calling into question the observations from the initial tors, leading to tumor regression in mouse models (77, shRNA screen (122). Regardless, inhibitors of CDK8 and, to 116). This was mediated, at least in part, by downregulating a lesser extent, CDK19 have been developed. Senexin A inhib-

MYC-driven transcription and correlated with preferential its CDK8 with an IC50 = 0.28 μM, although activity against downregulation of superenhancer-associated genes, indicat- other CDKs was not reported (123). This agent inhibits ing that CDK7 inhibitors can target the mechanisms that β-catenin transcriptional activity, and a derivative, Senexin B, promote global transcription amplification and might be a suppresses estrogen-dependent transcription in ER-positive useful strategy for targeting transcription factor oncopro- breast cancer (124). Other compounds targeting CDK8 and teins (116). THZ1 also inhibits androgen receptor signal- CDK19 have also been generated (122, 125), although as a ing in CRPC cells, reverses hyperphosphorylation of MED1 class, dual CDK8/19-targeting compounds may be too toxic (which is associated with a drug-resistant phenotype), and at therapeutic dose levels (126). Combinations of CDK8 induces tumor regression (117). Moreover, THZ1 and THZ2, inhibitors with drugs targeting orthogonal pathways, for an analogue with improved pharmacokinetic properties and example, glycolysis, may generate synergy and allow for more longer plasma half-life, were also efficacious in targeting tolerable levels of CDK8 inhibitors (119). Taken together, transcriptional addiction in TNBC cells (79). THZ2 inhib- although the evidence shows that CDK8 and CDK19 may be ited the growth of TNBC patient-derived xenograft (PDX) important targets, efforts to further develop these molecules models by targeting a cluster of transcription factor net- clinically will likely require improved targeting approaches works required for TNBC survival, including FOXC1, MYC, and a better understanding of whether pharmacologic inhibi- and SOX9. shRNA and CRISPR/Cas9–mediated silencing in tion will lead to cell death. SEL 120, a CDK8/19 inhibitor, is TNBC cells confirmed a high dependency onCDK7 as well as currently in early-phase clinical trials for hematologic malig- CDK9 but not CDK12 or CDK13 (79). In addition, chordoma nancies (Table 2). cell lines, which are a primary bone cancer with a dependency on transcription factor T (brachyury; TBXT) were sensitive CDK9 Inhibitors in vitro and in vivo to inhibitors targeting CDK7, as well as One of the first-generation CDK inhibitors, flavopiridol CDK9, CDK12, and CDK13 (118). Taken together, these (alvocidib), was a nonselective CDK inhibitor that most studies demonstrate that cancers dependent on oncogenic potently inhibited CDK9, with a reported Ki of 3 nM against transcription factors and their downstream networks can human cyclin T1-CDK9 in vitro (14, 127). In addition, fla- be therapeutically targeted by CDK7 inhibitors. Several tri- vopiridol also inhibited other transcription-associated als are currently testing CDK7 inhibitors in advanced solid CDKs including CDK7, as well as CDK1, CDK2, CDK4, and malignancies (Table 2). CDK6 with varying potencies (1). Despite promising in vitro activity, there was limited in vivo activity except in hema- CDK8/CDK19 Inhibitors tologic malignancies such as mantle cell lymphoma and The mediator-associated kinases CDK8 and CDK19 may CLL (1, 128, 129). In addition, nonselective CDK inhibitors also be important drug targets, although the oncogenic and suffer from narrow therapeutic windows causing adverse tumor-suppressive functions of CDK8 appear to be context- effects including bone marrow suppression, nausea, and dependent. In acute myeloid leukemia (AML), CDK8 gastrointestinal effects. Subsequently, development of fla- restrains key superenhancer-associated genes involved in vopiridol was halted. growth inhibition. Cortistatin A selectively inhibits CDK8 Dinaciclib (SCH727965) was initially developed as a more

(IC50 = 12 nM) and CDK19 (which are 94% identical in potent follow-up molecule to flavopiridol (130). It is a single- the catalytic domain), but not the other transcription- digit nanomolar inhibitor of CDK1, CDK2, CDK5, and associated CDKs (22). Inhibition of CDK8 and CDK19 CDK9 but also shows activity against CDK12 and CDK13. upregulates transcription of genes that inhibit AML cell Dinaciclib inhibits cell-cycle progression in >100 cell lines proliferation. Importantly, in leukemia mouse models, cor- tested (130) and clinical trials are ongoing, although prelimi- tistatin A has antitumor effects (22). CDK8 also plays a nary results are less encouraging than initially anticipated role in the hypoxic response and is a critical downstream (131). Interestingly, dinaciclib also promotes immunogenic

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Table 2. Current clinical trials utilizing transcription-associated CDK inhibitors in various cancer types

CDK inhibitor and Trial name target Disease types Objectives Phase I study of SY-1365, a selective CDK7 inhibi- SY-1365 (CDK7) Ovarian cancer Determine the DLTs, MTD, and tor, in adult patients with advanced solid tumors cohort, breast the safety and tolerability of (NCT03134638) cancer cohort, and SY-1365 as a single agent any advanced solid and in combination with either cancer cohort carboplatin or fulvestrant Modular, multipart, multiarm, open-label, phase I/ CT7001 (CDK7) Any advanced solid Determine the optimal mono- phase IIa study to evaluate the safety and toler- cancer therapy dose and combina- ability of CT7001 alone and in combination with tion doses of CT7001 and anticancer treatments in patients with advanced determine the safety and malignancies (NCT03363893) tolerability SEL120 in patients with AML or high-risk MDS SEL120 (CDK8/19) AML or high-risk MDS Determine the DLT, recommend- (NCT04021368) ed dosing, and MTD, tolerability, pharmacokinetics, and phamacodynamics of SEL120 Phase Ib/II, open-label clinical study to determine Alvocidib (CDK9) Myelodysplasia Determine the DLT and ORR preliminary safety and efficacy of alvocidib when based on IWG criteria administered in sequence after decitabine in patients with MDS (NCT03593915) Phase I, first-in-human, open-label, dose escalation, TP-1287 (CDK9) Any advanced solid Determine the DLT and MTD of safety, pharmacokinetic, and pharmacodynamic cancer TP-1287 study of oral TP-1287 to patients with advanced solid tumors (NCT03604783) Open-label, multicenter phase I study to character- BAY1251152 (CDK9) Any advanced hema- Determine the DLT and MTD as ize the safety, tolerability, preliminary antitumor tologic cancer well as phase II dose activity, pharmacokinetics and maximum tolerated dose of BAY1251152 in patients with advanced hematologic malignancies (NCT02745743) Completed An open-label, multicenter, two-stage, phase II study P276–00 (CDK9, Melanoma (stage IV) PFS at day 168 and OS at 1 year to evaluate efficacy and safety of P276–00 in CDK4 and CDK1) positive for cyclin subjects of malignant melanoma positive for D1 expression cyclin D1 expression (NCT00835419) Completed Study of TG02 in elderly newly diagnosed or adult TG02 (CDK1, CDK2, Anaplastic astrocyto- Determine the MTD and phase II relapsed patients with anaplastic astrocytoma or CDK7, CDK9) ma or glioblastoma dose, as well as PFS at glioblastoma: a phase Ib study (NCT03224104) 6 months Phase I, open-label, multicenter, nonrandomized AZD4573 (CDK9) Relapsed or refrac- Determine the DLT and MTD study to assess the safety, tolerability, pharma- tory advanced cokinetics and preliminary antitumor activity of hematologic cancer AZD4573, a potent and selective CDK9 inhibitor, in subjects with relapsed or refractory hematologic malignancies (NCT03263637) A phase I combination study of CYC065 and veneto- CYC065 (CDK2 and AML, MDS, and CLL Determine the MTD, analyze clax in patients with relapsed or refractory acute CDK9) pharmacokinetics and myeloid leukemia or MDSs (NCT04017546) and antitumor activity in relapsed or refractory chronic lymphocytic leukemia (NCT03739554) Phase I pharmacologic study of CYC065, a cyclin- CYC065 (CDK2 and Any advanced, meta- Determine the DLT and pharma- dependent kinase inhibitor, in patients with CDK9) static solid cancer cokinetics advanced cancers (NCT02552953) or lymphoma

Abbreviations: DLT, dose-limiting toxicity; MDS, myelodysplastic syndromes; IWG, International Working Group.

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REVIEW Chou et al. cell death and enhances anti–PD-1 checkpoint blockade by toxic chemotherapy regimens (104). In addition, CDK12 activating dendritic cells and increasing T-cell infiltrates, inhibitors might also have activity against another type of suggesting a potential rationale for combining dinaciclib sarcoma: osteosarcoma—a disease with limited therapeutic with anti–PD-1 checkpoint inhibitors (132). Additional pre- options (140). clinical and clinical studies will be required to determine A synthetic lethal interaction between CDK12 and MYC was whether this combination is tolerable and leads to improved previously demonstrated by Grandori and colleagues, who tumor control. performed a synthetic lethal siRNA screen of approximately The search for more specific CDK9 inhibitors continues, 3,300 druggable genes using fibroblasts overexpressingMYC , because it remains a promising target given its central role in and identified CDK12 as a synthetic lethal target (141). In transcription elongation (133). For example, CDK9 was iden- addition, ovarian cancer cells dependent on MYC are sensi- tified in a screen of more than 1,500 kinase inhibitors that tive to CDK12 inhibition. Treatment of 11 PDX models could specifically kill BRD4–NUT-rearranged NUT midline derived from patients with heavily pretreated ovarian cancer carcinoma (NMC) cells (134). NMC cells depend on CDK9 and with THZ1 suppressed tumor growth and abrogated MYC cyclin T1 expression, and their inhibition induces apoptosis. expression. Interestingly, MYC downregulation requires the A highly selective and potent CDK9 inhibitor (i-CDK9) was combined inhibition of CDK7, CDK12, and CDK13 in these recently identified that exhibits more than 600-fold selectiv- ovarian cancer models (142). In osteosarcoma, the sensitivity ity toward CDK9 (135). In addition, CDK9 inhibition with to CDK12 inhibitors may also be explained by MYC levels the kinase inhibitor PIK-75 in AML cells represses MCL1, (140). Targeting CDK12 with compound 919278 also inhibits a key AML survival factor. PIK-75, which also inhibits the osteosarcoma cells by transcriptionally regulating compo- p110α isoform of PI3K, reduces leukemia burden in vivo nents of the noncanonical NFκB signaling pathway (143). (136). Pharmacologic strategies to degrade CDK9 have also Taken together, these data support the notion that in cancers been explored. These compounds consist of a CDK-binding dependent on certain transcription factors (i.e., MYC, EWS– ligand (SNS-032) linked to a thalidomide derivative that FLI1, NFkB), targeting CDK12 and other transcription-asso- binds to the E3 ubiquitin ligase Cereblon to promote deg- ciated CDKs may be a viable strategy. However, specifically radation (137). Finally, a highly selective CDK9 inhibitor, targeting CDK12 has been challenging due to the similarities

MC180295 (IC50 of 5 nM), was recently shown to have broad with other CDKs, especially CDK13; the kinase domains of anticancer effects in vitro and in vivo. Interestingly, the mech- CDK12 and CDK13 are 92% identical, making it difficult to anism of tumor suppression was not attributed to inhibi- generate specific CDK12 or CDK13 kinase inhibitors through tion of transcription elongation, but instead to reactivating conventional routes. epigenetically silenced genes, thereby restoring tumor sup- THZ531 is a THZ1 derivative with 50-fold greater pressor gene expression and cell differentiation (138). CDK9 potency against CDK12/CDK13 than CDK7 or CDK9 (e.g., inhibition decreased phosphorylation of the SWI/SNF pro- CDK12 IC50 = 158 nM vs. CDK7 IC50 = 8,500 nM; ref. 60). tein BRG1, and led to genome-wide epigenetic derepression, However, THZ531 is subject to drug efflux through upregu- suggesting that CDK9 is a novel epigenetic target (138). lation of the ABCB1 and ABCG2 transporters, a potential Moreover, CDK9 inhibition also activated an IFN response, resistance mechanism (144). THZ531 covalently binds the as well as endogenous retroviruses and increased sensitivity cysteine residue at position 1039 as well as CDK13 at to immune checkpoint blockade (138). Several clinical trials cysteine 1017 and CDK7 at cysteine 312. Compound E9 testing CDK9 inhibitors are currently being conducted in was developed to overcome resistance to the THZ series of advanced solid and hematologic malignancies (Table 2). As transcriptional CDK inhibitors by protecting it from drug CDK9 inhibition may reactivate tumor-suppressor genes and efflux (144). Compound E9 also targets cysteine residues induce cellular immune responses that increase sensitivity to in CDK7/12/13, and so cells harboring a C1039S mutation checkpoint inhibition, the development of additional, more in CDK12 are resistant to compound E9 (Supplementary specific CDK9 inhibitors and combination therapies will be Table S1). of significant interest. Ito and colleagues (Takeda) recently developed Com- pound 2, in an effort to derive a CDK12 inhibitor with higher specificity and inhibitory properties as well as physi- CDK12/CDK13 Inhibitors ochemical properties (145). This compound inhibits Ser2 In addition, CDK12 has also attracted considerable atten- phosphorylation in the CTD of RNA Pol II and growth of tion as a drug target. CDK12 is critical in Ewing sarcoma, SK-BR-3 breast cancer cells, which overexpress HER2. In which is sensitive to CDK12 inhibitors and appears to addition, inhibition of CDK12 enhances HER2-targeting synergize with PARPi (139). The tumor-specific expression therapies, and HER2-amplified tumors may be sensitive to of the EWS–FLI oncofusion mediates the sensitivity to the CDK12 inhibitors (146). Johannes and colleagues (Astra- combination. Although the mechanism for this sensitivity Zeneca) used structure-based design to develop noncova- remains to be fully elucidated, it may relate to the preexist- lent inhibitors of CDK12 with moderate potency (147). ing sensitivity of Ewing sarcoma to DNA-damaging agents Their data suggest that the window of inhibition with such as doxorubicin and etoposide, which are used to treat CDK12 may be too small to achieve a therapeutic index Ewing sarcoma. It remains to be seen whether simultane- in the tumor over normal tissue. Surprisingly, they did ous, systemic suppression of CDK12 and PARP is a tolerable not observe any synergy with PARPi; the explanation for combination in patients; bone marrow toxicity has been a this is currently unclear. In contrast, SR-4835, an ATP- significant issue when combining many PARPi with cyto- competitive kinase inhibitor against CDK12 and CDK13,

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Transcription-Associated CDKs in Cancer REVIEW has been shown to suppress the expression of multiple nals (155). These data suggest that inhibitors against the DNA-repair genes and regulate intronic polyadenylation, transcription-associated CDKs may have broad indications which increases sensitization to platinum chemotherapy in human disease. and PARPi in a model of TNBC (148). Although several research-grade tool compounds are currently available (Supplementary Table S1), no clinical-grade CDK12 or CONCLUSIONS, FUTURE PROSPECTS, AND CDK13 inhibitor is yet available. KEY QUESTIONS The transcription-associated CDKs are emerging as important targets and biomarkers in oncology. In contrast to Targeting Therapeutic Resistance the cell cycle–associated CDKs, this family of CDKs plays The ability to suppress adaptive responses to targeted can- critical roles in regulating gene expression at multiple lev- cer therapies by repressing transcription has been proposed, els, including transcription, splicing, intronic polyadenyla- as therapy resistance remains a major hurdle. Resistance tion, and epigenetics. Recent evidence also points to a role appears to be at least partially due to the acquisition of adap- in regulating protein expression at the level of translation, tive cellular programs. Inhibition of CDK7/CDK12/CDK13 although additional work will be required to sort out whether with THZ1 blocks transcriptional programs that facilitate this is an indirect effect, and whether as a family the tran- resistance. Although the exact mechanism remains to be scription-associated CDKs affect these processes. How these fully elucidated, it may involve remodeling of enhancers and CDKs interact with each other to regulate complex molecular other signaling outputs required for tumor cell survival in processes also remains to be more fully elucidated. What the setting of targeted therapy. Accordingly, genetic ablation intrinsic redundancies are in place, and how do these CDKs of either CDK7 or CDK12 reduces the outgrowth of resistant work together to affect cancer cell properties such as growth, clones (149). THZ531 also impairs the emergence of resist- invasion, and apoptosis? These answers will likely be cell con- ance. Furthermore, acquired resistance to BET inhibitors in text–dependent, and so understanding which cells rely on CRPC results in reactivation of androgen receptor signaling which particular transcription-associated CDKs will be critical. (150). This is mediated by CDK9-mediated androgen recep- Loss of particular transcription-associated CDKs or their tor phosphorylation, as inhibition of CDK9 with dinaciclib cyclin partners may also uncover potential therapeutic vul- or LDC000067 suppresses androgen receptor and its tran- nerabilities and synthetic lethal interactions. To date, most scriptional activity in BET inhibitor–resistant cells (150). transcriptional CDKs are not frequently mutated or lost, with The addition of transcriptional CDK inhibitors to targeted the exception of CDK12. Preclinical evidence demonstrates cancer therapies may therefore delay resistance and lead to that CDK12 deficiency increases sensitivity to PARP and CHK1 more durable responses, although more robust preclinical inhibitors, and, because of the genomic instability that results work needs to be done prior to testing these combinations from CDK12 loss, may also increase susceptibility to check- in clinical trials. point immunotherapy. However, our mechanistic under- Resistance to PARPi also remains an important limita- standing of this phenomenon and the precise role CDK12 tion, even in patients with BRCA1/BRCA2-mutated cancer plays in DNA replication and TDs remains poor. Nonetheless, (104, 151, 152). CDK12 inhibitors might reverse de novo and several of these targeted strategies are currently being tested acquired PARPi resistance in BRCA1-mutant breast cancer in clinical trials, particularly in advanced prostate cancer. Yet cells (153). Dinaciclib (which inhibits CDK9, CDK12, and our understanding of how specificCDK12 mutations affect CDK13 as well as CDK1, CDK2, and CDK5) reduces levels their function remains limited, and how other molecular of BRCA1 and RAD51 mRNAs and levels of HR DNA repair. alterations occur in context (whether in cis or in trans) will be This sensitizes BRCA1-mutant and wild-type TNBC cells important to work out, which will likely affect the efficacy of to PARPi, which can be phenocopied by genetic CDK12 these targeted approaches. depletion. In models of PARPi resistance, CDK12 inhibi- Additional work on developing more specific chemical tion restores PARPi sensitivity (153). Whether more specific inhibitors that target the transcription-associated CDKs will CDK12 inhibitors will be efficacious in treating or delaying also be important, as the side-effect profiles of these inhibi- PARPi resistance, and whether the combination of CDK12 tors may limit patient tolerability. Several studies point to inhibitors and PARPi is tolerable in patients remains to be the use of CDK7, CDK8, CDK9, CDK12, and CDK13 inhibi- determined. tors in various solid and hematologic cancer types, including TNBC, MYCN-driven neuroblastoma, colon cancer, Ewing Targeting Transcription-Associated CDKs sarcoma, AML, and SCLC. These cancers are primarily driven beyond Cancer by dysregulated transcription, and are highly dependent on Beyond oncology, transcription-associated kinase inhibi- high levels of basal transcription mediated through transcrip- tors may also find indications in other disease areas, includ- tion factors such as MYC, β-catenin, or EWS–FLI1. Several ing infectious diseases and cardiology (14). Indeed, a recent clinical trials with CDK7 and CDK9 inhibitors are currently study showed that a novel compound active against visceral under way to determine the MTD and dose-limiting toxici- leishmaniasis acts principally by inhibiting CDC2-related ties, to determine whether these can be successfully admin- kinase 12 (CRK12), the Leishmania homolog of CDK12 istered to patients. In addition, for WNT-driven tumors and (154). In addition, cardiac hypertrophy is characterized those harboring CDK8 amplification, CDK8 inhibition may by global increases in mRNA and protein synthesis, and hold therapeutic promise, but this awaits to be tested clini- inactivation of Cdk9 in mice dampens hypertrophic sig- cally. Moreover, these transcriptional CDK inhibitors may

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REVIEW Chou et al. delay adaptive resistance to targeted therapies, including 6. Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regu- to PARPi. Therefore, combination strategies will likely be lation. Development 2013;140:3079–93. important, including with immunotherapy, although the 7. Serizawa H, Makela TP, Conaway JW, Conaway RC, Weinberg RA, Young RA. Association of Cdk-activating kinase subunits with tran- therapeutic index and cumulative toxicities will need to be scription factor TFIIH. Nature 1995;374:280–2. carefully monitored. Finally, inhibitors of other lesser-known 8. Spangler L, Wang X, Conaway JW, Conaway RC, Dvir A. TFIIH transcription-associated CDKs, such as CDK11, may also be action in transcription initiation and promoter escape requires important targets in cancer (156), and underscore the need distinct regions of downstream promoter DNA. Proc Natl Acad Sci for additional research beyond the canonical transcription- U S A 2001;98:5544–9. associated CDKs. 9. Larochelle S, Amat R, Glover-Cutter K, Sanso M, Zhang C, Allen JJ, et al. Cyclin-dependent kinase control of the initiation-to-elonga- In summary, understanding the transcription-associated tion switch of RNA polymerase II. Nat Struct Mol Biol 2012;19: CDKs is an exciting area of great promise in oncology, one 1108–15. that will likely continue to receive considerable attention in 10. Fisher RP. Cdk7: a kinase at the core of transcription and in the coming years. We eagerly await the entry of transcription- the crosshairs of cancer drug discovery. Transcription 2019;10: associated CDK inhibitors into the clinical armamentarium 47–56. against cancer, and anticipate the identification of new syn- 11. Rimel JK, Taatjes DJ. The essential and multifunctional TFIIH com- thetic lethal strategies to target tumors characterized by the plex. Protein Sci 2018;27:1018–37. 12. Fu TJ, Peng J, Lee G, Price DH, Flores O. Cyclin K functions as a loss of these important regulators. CDK9 regulatory subunit and participates in RNA polymerase II transcription. J Biol Chem 1999;274:34527–30. Disclosure of Potential Conflicts of Interest 13. Peng J, Zhu Y, Milton JT, Price DH. Identification of multiple cyclin F.Y. Feng has paid consultant/advisory board relationships with subunits of human P-TEFb. Genes Dev 1998;12:755–62. Janssen, Sanofi, Astellas, Bayer, Genentech, EMD Serono, Clovis, and 14. Wang S, Fischer PM. Cyclin-dependent kinase 9: a key transcrip- Celgene, and an unpaid consultant/advisory board relationship with tional regulator and potential drug target in oncology, virology and PFS Genomics. A. Ashworth is a cofounder of Tango Therapeutics, cardiology. Trends Pharmacol Sci 2008;29:302–13. Azkarra Therapeutics, and Ovibio, is an advisor for Gladiator, Pro- 15. Herrmann CH, Mancini MA. The Cdk9 and cyclin T subunits of lynx, Earli, and Genentech, reports receiving commercial research TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions. grants from AstraZeneca and SPARC, and has ownership interest in J Cell Sci 2001;114:1491–503. patents on the use of PARP inhibitors, held jointly with AstraZen- 16. Yu DS, Zhao R, Hsu EL, Cayer J, Ye F, Guo Y, et al. Cyclin-dependent eca. No potential conflicts of interest were disclosed by the other kinase 9-cyclin K functions in the replication stress response. EMBO Rep 2010;11:876–82. authors. 17. Lolli G, Lowe ED, Brown NR, Johnson LN. The crystal structure of human CDK7 and its protein recognition properties. Structure Acknowledgments 2004;12:2067–79. J. Chou is supported by the A.P. Giannini Foundation, the Quinlan 18. Tassan JP, Jaquenoud M, Leopold P, Schultz SJ, Nigg EA. Identifica- and Rosenberg Fellowships in Genitourinary Oncology, and a train- tion of human cyclin-dependent kinase 8, a putative protein kinase ing grant from the NCI (T32 CA108462). D.A. Quigley is supported partner for cyclin C. Proc Natl Acad Sci U S A 1995;92:8871–5. by Young Investigator awards from the Prostate Cancer Foundation 19. Flanagan PM, Kelleher RJ III, Sayre MH, Tschochner H, Kornberg and the BRCA Foundation. F.Y. Feng and A. Ashworth are supported RD. A mediator required for activation of RNA polymerase II tran- by Challenge Awards from the Prostate Cancer Foundation. This scription in vitro. Nature 1991;350:436–8. work was supported by the NCI (1R01CA230516; to F.Y. Feng and 20. Allen BL, Taatjes DJ. The Mediator complex: a central integrator of A. Ashworth). transcription. Nat Rev Mol Cell Biol 2015;16:155–66. 21. Akoulitchev S, Chuikov S, Reinberg D. TFIIH is negatively regu- lated by cdk8-containing mediator complexes. Nature 2000;407: Received May 14, 2019; revised September 29, 2019; accepted 102–6. November 4, 2019; published first February 18, 2020. 22. Pelish HE, Liau BB, Nitulescu II, Tangpeerachaikul A, Poss ZC, Da Silva DH, et al. Mediator kinase inhibition further activates superenhancer-associated genes in AML. Nature 2015;526:273–6. 23. Galbraith MD, Allen MA, Bensard CL, Wang X, Schwinn MK, Qin B, REFERENCES et al. HIF1A employs CDK8-mediator to stimulate RNAPII elonga- . 1 Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. The history and tion in response to hypoxia. Cell 2013;153:1327–39. future of targeting cyclin-dependent kinases in cancer therapy. Nat 24. Donner AJ, Ebmeier CC, Taatjes DJ, Espinosa JM. CDK8 is a positive Rev Drug Discov 2015;14:130–46. regulator of transcriptional elongation within the serum response 2. Hortobagyi GN, Stemmer SM, Burris HA, Yap YS, Sonke GS, network. Nat Struct Mol Biol 2010;17:194–201. Paluch-Shimon S, et al. Ribociclib as first-line therapy for HR- 25. Fant CB, Taatjes DJ. Regulatory functions of the mediator kinases positive, advanced breast cancer. N Engl J Med 2016;375:1738–48. CDK8 and CDK19. Transcription 2019;10:76–90. 3. Finn RS, Martin M, Rugo HS, Jones S, Im SA, Gelmon K, et al. 26. Chen M, Liang J, Ji H, Yang Z, Altilia S, Hu B, et al. CDK8/19 media- Palbociclib and letrozole in advanced breast cancer. N Engl J Med tor kinases potentiate induction of transcription by NFkappaB. 2016;375:1925–36. Proc Natl Acad Sci U S A 2017;114:10208–13. 4. Sledge GW Jr, Toi M, Neven P, Sohn J, Inoue K, Pivot X, et al. 27. Malumbres M. Cyclin-dependent kinases. Genome Biol 2014;15:122. MONARCH 2: abemaciclib in combination with fulvestrant in 28. Audetat KA, Galbraith MD, Odell AT, Lee T, Pandey A, Espinosa JM, women with HR+/HER2- advanced breast cancer who had progressed et al. A kinase-independent role for cyclin-dependent Kinase 19 in while receiving endocrine therapy. J Clin Oncol 2017;35:2875–84. p53 response. Mol Cell Biol 2017;37:pii:e00626–16. 5. Kumar SK, LaPlant B, Chng WJ, Zonder J, Callander N, Fonseca R, 29. Poss ZC, Ebmeier CC, Odell AT, Tangpeerachaikul A, Lee T, Pelish et al. Dinaciclib, a novel CDK inhibitor, demonstrates encouraging HE, et al. Identification of mediator kinase substrates in human single-agent activity in patients with relapsed multiple myeloma. cells using cortistatin a and quantitative phosphoproteomics. Cell Blood 2015;125:443–8. Rep 2016;15:436–50.

OF16 | CANCER DISCOVERY March 2020 AACRJournals.org

Transcription-Associated CDKs in Cancer REVIEW

30. Ko TK, Kelly E, Pines J. CrkRS: a novel conserved Cdc2-related 52. Moreland RJ, Tirode F, Yan Q, Conaway JW, Egly JM, Conaway RC. protein kinase that colocalises with SC35 speckles. J Cell Sci 2001; A role for the TFIIH XPB DNA helicase in promoter escape by RNA 114:2591–603. polymerase II. J Biol Chem 1999;274:22127–30. 31. Juan HC, Lin Y, Chen HR, Fann MJ. Cdk12 is essential for embry- 53. Wong KH, Jin Y, Struhl K. TFIIH phosphorylation of the Pol II CTD onic development and the maintenance of genomic stability. Cell stimulates mediator dissociation from the preinitiation complex Death Differ 2016;23:1038–48. and promoter escape. Mol Cell 2014;54:601–12. 32. Chen HH, Wang YC, Fann MJ. Identification and characterization 54. Olson CM, Liang Y, Leggett A, Park WD, Li L, Mills CE, et al. Devel- of the CDK12/cyclin L1 complex involved in alternative splicing opment of a selective CDK7 covalent inhibitor reveals predominant regulation. Mol Cell Biol 2006;26:2736–45. cell-cycle phenotype. Cell Chem Biol 2019;26:792–803. 33. Bartkowiak B, Liu P, Phatnani HP, Fuda NJ, Cooper JJ, Price DH, 55. Coin F, Egly JM. Revisiting the function of CDK7 in transcription et al. CDK12 is a transcription elongation-associated CTD by virtue of a recently described TFIIH kinase inhibitor. Mol Cell kinase, the metazoan ortholog of yeast Ctk1. Genes Dev 2010;24: 2015;59:513–4. 2303–16. 56. Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, et al. Recruitment 34. Cheng SW, Kuzyk MA, Moradian A, Ichu TA, Chang VC, Tien JF, of P-TEFb for stimulation of transcriptional elongation by the bro- et al. Interaction of cyclin-dependent kinase 12/CrkRS with cyclin modomain protein Brd4. Mol Cell 2005;19:535–45. K1 is required for the phosphorylation of the C-terminal domain of 57. Galbraith MD, Bender H, Espinosa JM. Therapeutic targeting of RNA polymerase II. Mol Cell Biol 2012;32:4691–704. transcriptional cyclin-dependent kinases. Transcription 2018;10: 35. Bosken CA, Farnung L, Hintermair C, Merzel Schachter M, Vogel- 118–36. Bachmayr K, Blazek D, et al. The structure and substrate specificity 58. Xie Q, Wu Q, Kim L, Miller TE, Liau BB, Mack SC, et al. RBPJ of human Cdk12/Cyclin K. Nat Commun 2014;5:3505. maintains brain tumor-initiating cells through CDK9-mediated 36. Kohoutek J, Blazek D. Cyclin K goes with Cdk12 and Cdk13. Cell transcriptional elongation. J Clin Invest 2016;126:2757–72. Div 2012;7:12. 59. Sanso M, Levin RS, Lipp JJ, Wang VY, Greifenberg AK, Quezada EM, 37. Greifenberg AK, Honig D, Pilarova K, Duster R, Bartholomeeusen K, et al. P-TEFb regulation of transcription termination factor Xrn2 Bosken CA, et al. Structural and functional analysis of the Cdk13/ revealed by a chemical genetic screen for Cdk9 substrates. Genes Cyclin K complex. Cell Rep 2016;14:320–31. Dev 2016;30:117–31. 38. Liang K, Gao X, Gilmore JM, Florens L, Washburn MP, Smith E, 60. Zhang T, Kwiatkowski N, Olson CM, Dixon-Clarke SE, Abraham et al. Characterization of human cyclin-dependent kinase 12 (CDK12) BJ, Greifenberg AK, et al. Covalent targeting of remote cysteine and CDK13 complexes in C-terminal domain phosphorylation, residues to develop CDK12 and CDK13 inhibitors. Nat Chem Biol gene transcription, and RNA processing. Mol Cell Biol 2015;35: 2016;12:876–84. 928–38. 61. Chirackal Manavalan AP, Pilarova K, Kluge M, Bartholomeeusen 39. Whittaker SR, Mallinger A, Workman P, Clarke PA. Inhibitors of K, Rajecky M, Oppelt J, et al. CDK12 controls G1/S progression cyclin-dependent kinases as cancer therapeutics. Pharmacol Ther by regulating RNAPII processivity at core DNA replication genes. 2017;173:83–105. EMBO Rep 2019;20:e47592. 40. Loyer P, Trembley JH, Grenet JA, Busson A, Corlu A, Zhao W, et al. 62. Dubbury SJ, Boutz PL, Sharp PA. CDK12 regulates DNA repair genes Characterization of cyclin L1 and L2 interactions with CDK11 and by suppressing intronic polyadenylation. Nature 2018;564:141–5. splicing factors: influence of cyclin L isoforms on splice site selec- 63. Krajewska M, Dries R, Grassetti AV, Dust S, Gao Y, Huang H, et al. tion. J Biol Chem 2008;283:7721–32. CDK12 loss in cancer cells affects DNA damage response genes 41. Hu D, Mayeda A, Trembley JH, Lahti JM, Kidd VJ. CDK11 complexes through premature cleavage and polyadenylation. Nat Commun promote pre-mRNA splicing. J Biol Chem 2003;278:8623–9. 2019;10:1757. 42. Drogat J, Migeot V, Mommaerts E, Mullier C, Dieu M, van Bakel H, 64. Blazek D, Kohoutek J, Bartholomeeusen K, Johansen E, Hulinkova et al. Cdk11-cyclinL controls the assembly of the RNA polymerase P, Luo Z, et al. The Cyclin K/Cdk12 complex maintains genomic sta- II mediator complex. Cell Rep 2012;2:1068–76. bility via regulation of expression of DNA damage response genes. 43. Guen VJ, Gamble C, Flajolet M, Unger S, Thollet A, Ferandin Y, Genes Dev 2011;25:2158–72. et al. CDK10/cyclin M is a protein kinase that controls ETS2 degra- 65. Ekumi KM, Paculova H, Lenasi T, Pospichalova V, Bosken CA, Ryba- dation and is deficient in STAR syndrome. Proc Natl Acad Sci U S A rikova J, et al. Ovarian carcinoma CDK12 mutations misregulate 2013;110:19525–30. expression of DNA repair genes via deficient formation and function 44. Iorns E, Turner NC, Elliott R, Syed N, Garrone O, Gasco M, et al. of the Cdk12/CycK complex. Nucleic Acids Res 2015;43:2575–89. Identification of CDK10 as an important determinant of resist- 66. Chen HR, Lin GT, Huang CK, Fann MJ. Cdk12 and Cdk13 regu- ance to endocrine therapy for breast cancer. Cancer Cell 2008;13: late axonal elongation through a common signaling pathway that 91–104. modulates Cdk5 expression. Exp Neurol 2014;261:10–21. 45. Zhou Y, Han C, Li D, Yu Z, Li F, Li F, et al. Cyclin-dependent kinase 67. Li X, Chatterjee N, Spirohn K, Boutros M, Bohmann D. Cdk12 is 11(p110) (CDK11(p110)) is crucial for human breast cancer cell a gene-selective RNA polymerase II kinase that regulates a subset proliferation and growth. Sci Rep 2015;5:10433. of the transcriptome, including Nrf2 target genes. Sci Rep 2016; 46. Jia B, Choy E, Cote G, Harmon D, Ye S, Kan Q, et al. Cyclin-depend- 6:21455. ent kinase 11 (CDK11) is crucial in the growth of liposarcoma cells. 68. Eifler TT, Shao W, Bartholomeeusen K, Fujinaga K, Jager S, Johnson Cancer Lett 2014;342:104–12. JR, et al. Cyclin-dependent kinase 12 increases 3′ end processing of 47. Rosonina E, Blencowe BJ. Analysis of the requirement for RNA growth factor-induced c-FOS transcripts. Mol Cell Biol 2015;35: polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 468–78. 3′-end cleavage. RNA 2004;10:581–9. 69. Even Y, Escande ML, Fayet C, Geneviere AM. CDK13, a kinase 48. Chen FX, Smith ER, Shilatifard A. Born to run: control of tran- involved in pre-mRNA splicing, is a component of the perinucleolar scription elongation by RNA polymerase II. Nat Rev Mol Cell Biol compartment. PLoS One 2016;11:e0149184. 2018;19:464–78. 70. Tien JF, Mazloomian A, Cheng SG, Hughes CS, Chow CCT, Canapi 49. Sainsbury S, Bernecky C, Cramer P. Structural basis of transcription LT, et al. CDK12 regulates alternative last exon mRNA splicing and initiation by RNA polymerase II. Nat Rev Mol Cell Biol 2015;16:129–43. promotes breast cancer cell invasion. Nucleic Acids Res 2017;45: 50. Asturias FJ. RNA polymerase II structure, and organization of the 6698–716. preinitiation complex. Curr Opin Struct Biol 2004;14:121–9. 71. Elkon R, Ugalde AP, Agami R. Alternative cleavage and polyade- 51. Compe E, Egly JM. TFIIH: when transcription met DNA repair. Nat nylation: extent, regulation and function. Nat Rev Genet 2013;14: Rev Mol Cell Biol 2012;13:343–54. 496–506.

march 2020 CANCER DISCOVERY | OF17

REVIEW Chou et al.

72. Lee SH, Singh I, Tisdale S, Abdel-Wahab O, Leslie CS, Mayr C. 93. Popova T, Manie E, Boeva V, Battistella A, Goundiam O, Smith NK, Widespread intronic polyadenylation inactivates tumour suppres- et al. Ovarian cancers harboring inactivating mutations in CDK12 sor genes in leukaemia. Nature 2018;561:127–31. display a distinct genomic instability pattern characterized by large 73. Pan L, Xie W, Li KL, Yang Z, Xu J, Zhang W, et al. Heterochromatin tandem duplications. Cancer Res 2016;76:1882–91. remodeling by CDK12 contributes to learning in Drosophila. Proc 94. Menghi F, Barthel FP, Yadav V, Tang M, Ji B, Tang Z, et al. The Natl Acad Sci U S A 2015;112:13988–93. tandem duplicator phenotype is a prevalent genome-wide cancer 74. Ebmeier CC, Erickson B, Allen BL, Allen MA, Kim H, Fong N, configuration driven by distinct gene mutations. Cancer Cell 2018; et al. Human TFIIH kinase CDK7 regulates transcription-associated 34:197–210. chromatin modifications. Cell Rep 2017;20:1173–86. 95. Cancer Genome Atlas Research Network. The molecular taxonomy 75. Edmunds JW, Mahadevan LC, Clayton AL. Dynamic histone H3 of primary prostate cancer. Cell 2015;163:1011–25. methylation during gene induction: HYPB/Setd2 mediates all 96. Robinson D, Van Allen EM, Wu YM, Schultz N, Lonigro RJ, Mos- H3K36 trimethylation. EMBO J 2008;27:406–20. quera JM, et al. Integrative clinical genomics of advanced prostate 76. Choi SH, Martinez TF, Kim S, Donaldson C, Shokhirev MN, Saghat- cancer. Cell 2015;162:454. elian A, et al. CDK12 phosphorylates 4E-BP1 to enable mTORC1- 97. Reimers MA, Yip SM, Zhang L, Cieslik M, Dhawan M, Montgomery dependent translation and mitotic genome stability. Genes Dev B, et al. Clinical outcomes in cyclin-dependent kinase 12 mutant 2019;33:418–35. advanced prostate cancer. Eur Urol 2019 Oct 20 [Epub ahead of 77. Chipumuro E, Marco E, Christensen CL, Kwiatkowski N, Zhang T, print]. Hatheway CM, et al. CDK7 inhibition suppresses super-enhancer- 98. Wu YM, Cieslik M, Lonigro RJ, Vats P, Reimers MA, Cao X, et al. linked oncogenic transcription in MYCN-driven cancer. Cell Inactivation of CDK12 delineates a distinct immunogenic class of 2014;159:1126–39. advanced prostate cancer. Cell 2018;173:1770–82. 78. Firestein R, Bass AJ, Kim SY, Dunn IF, Silver SJ, Guney I, et al. CDK8 99. Nguyen VQ, Co C, Li JJ. Cyclin-dependent kinases prevent DNA is a colorectal cancer oncogene that regulates beta-catenin activity. re-replication through multiple mechanisms. Nature 2001;411: Nature 2008;455:547–51. 1068–73. 79. Wang Y, Zhang T, Kwiatkowski N, Abraham BJ, Lee TI, Xie S, et al. 100. Green BM, Finn KJ, Li JJ. Loss of DNA replication control is a potent CDK7-dependent transcriptional addiction in triple-negative breast inducer of gene amplification. Science 2010;329:943–6. cancer. Cell 2015;163:174–86. 101. Lei T, Zhang P, Zhang X, Xiao X, Zhang J, Qiu T, et al. Cyclin K 80. Naidoo K, Wai PT, Maguire SL, Daley F, Haider S, Kriplani D, regulates prereplicative complex assembly to promote mammalian et al. Evaluation of CDK12 protein expression as a potential novel cell proliferation. Nat Commun 2018;9:1876. biomarker for DNA damage response-targeted therapies in breast 102. Willis NA, Frock RL, Menghi F, Duffey EE, Panday A, Camacho V, cancer. Mol Cancer Ther 2018;17:306–15. et al. Mechanism of tandem duplication formation in BRCA1- 81. Li H, Lahti JM, Valentine M, Saito M, Reed SI, Look AT, et al. Molec- mutant cells. Nature 2017;551:590–5. ular cloning and chromosomal localization of the human cyclin C 103. Menghi F, Inaki K, Woo X, Kumar PA, Grzeda KR, Malhotra A, et al. (CCNC) and cyclin E (CCNE) genes: deletion of the CCNC gene in The tandem duplicator phenotype as a distinct genomic configura- human tumors. Genomics 1996;32:253–9. tion in cancer. Proc Natl Acad Sci U S A 2016;113:E2373–82. 82. Li N, Fassl A, Chick J, Inuzuka H, Li X, Mansour MR, et al. Cyclin 104. Lord CJ, Ashworth A. PARP inhibitors: synthetic lethality in the C is a haploinsufficient tumour suppressor. Nat Cell Biol 2014;16: clinic. Science 2017;355:1152–8. 1080–91. 105. Ashworth A, Lord CJ. Synthetic lethal therapies for cancer: what’s 83. Cancer Genome Atlas Research Network. Integrated genomic analy- next after PARP inhibitors? Nat Rev Clin Oncol 2018;15:564–76. ses of ovarian carcinoma. Nature 2011;474:609–15. 106. Joshi PM, Sutor SL, Huntoon CJ, Karnitz LM. Ovarian cancer- 84. Bajrami I, Frankum JR, Konde A, Miller RE, Rehman FL, Brough R, associated mutations disable catalytic activity of CDK12, a kinase et al. Genome-wide profiling of genetic synthetic lethality identifies that promotes homologous recombination repair and resistance to CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity. cisplatin and poly(ADP-ribose) polymerase inhibitors. J Biol Chem Cancer Res 2014;74:287–97. 2014;289:9247–53. 85. Carter SL, Cibulskis K, Helman E, McKenna A, Shen H, Zack T, et al. 107. Paculova H, Kramara J, Simeckova S, Fedr R, Soucek K, Hylse O, Absolute quantification of somatic DNA alterations in human can- et al. BRCA1 or CDK12 loss sensitizes cells to CHK1 inhibitors. cer. Nat Biotechnol 2012;30:413–21. Tumour Biol 2017;39:1010428317727479. 86. Riaz N, Blecua P, Lim RS, Shen R, Higginson DS, Weinhold N, et al. 108. Schiewer MJ, Goodwin JF, Han S, Brenner JC, Augello MA, Dean JL, Pan-cancer analysis of bi-allelic alterations in homologous recombi- et al. Dual roles of PARP-1 promote cancer growth and progression. nation DNA repair genes. Nat Commun 2017;8:857. Cancer Discov 2012;2:1134–49. 87. Quigley DA, Dang HX, Zhao SG, Lloyd P, Aggarwal R, Alumkal JJ, 109. Paculova H, Kohoutek J. The emerging roles of CDK12 in tumori- et al. Genomic hallmarks and structural variation in metastatic genesis. Cell Div 2017;12:7. prostate cancer. Cell 2018;174:758–69. 110. Mertins P, Mani DR, Ruggles KV, Gillette MA, Clauser KR, Wang P, 88. Viswanathan SR, Ha G, Hoff AM, Wala JA, Carrot-Zhang J, Whelan et al. Proteogenomics connects somatic mutations to signalling in CW, et al. Structural alterations driving castration-resistant pros- breast cancer. Nature 2016;534:55–62. tate cancer revealed by linked-read genome sequencing. Cell 2018; 111. Natrajan R, Wilkerson PM, Marchio C, Piscuoglio S, Ng CK, Wai 174:433–47. P, et al. Characterization of the genomic features and expressed 89. Stephens PJ, McBride DJ, Lin ML, Varela I, Pleasance ED, Simpson fusion genes in micropapillary carcinomas of the breast. J Pathol JT, et al. Complex landscapes of somatic rearrangement in human 2014;232:553–65. breast cancer genomes. Nature 2009;462:1005–10. 112. Abida W, Bryce AH, Vogelzang NJ, Amatao RJ, Percent I, Shap- 90. Ng CK, Cooke SL, Howe K, Newman S, Xian J, Temple J, et al. The iro J, et al. Preliminary results from TRITON2: a phase 2 study role of tandem duplicator phenotype in tumour evolution in high- of rucaparib in patients with metastatic castration-resistant pros- grade serous ovarian cancer. J Pathol 2012;226:703–12. tate cancer (mCRPC) associated with homologous recombination 91. McBride DJ, Etemadmoghadam D, Cooke SL, Alsop K, George repair (HRR) gene alterations. Ann Oncol 2018;29(suppl_8):viii271– J, Butler A, et al. Tandem duplication of chromosomal segments viii302. is common in ovarian and breast cancer genomes. J Pathol 2012; 113. Antonarakis ES. Cyclin-Dependent Kinase 12, immunity, and pros- 227:446–55. tate cancer. N Engl J Med 2018;379:1087–9. 92. Nik-Zainal S, Davies H, Staaf J, Ramakrishna M, Glodzik D, Zou X, 114. Kotsantis P, Silva LM, Irmscher S, Jones RM, Folkes L, Gromak N, et al. Landscape of somatic mutations in 560 breast cancer whole- et al. Increased global transcription activity as a mechanism of genome sequences. Nature 2016;534:47–54. replication stress in cancer. Nat Commun 2016;7:13087.

OF18 | CANCER DISCOVERY March 2020 AACRJournals.org

Transcription-Associated CDKs in Cancer REVIEW

115. Kwiatkowski N, Zhang T, Rahl PB, Abraham BJ, Reddy J, Ficarro SB, CDK9 as a synthetic lethal target in NUT midline carcinoma. Cell et al. Targeting transcription regulation in cancer with a covalent Rep 2017;20:2833–45. CDK7 inhibitor. Nature 2014;511:616–20. 135. Lu H, Xue Y, Yu GK, Arias C, Lin J, Fong S, et al. Compensatory 116. Christensen CL, Kwiatkowski N, Abraham BJ, Carretero J, Al-Shah- induction of MYC expression by sustained CDK9 inhibition via a rour F, Zhang T, et al. Targeting transcriptional addictions in BRD4-dependent mechanism. Elife 2015;4:e06535. small cell lung cancer with a covalent CDK7 inhibitor. Cancer Cell 136. Thomas D, Powell JA, Vergez F, Segal DH, Nguyen NY, Baker A, 2014;26:909–22. et al. Targeting acute myeloid leukemia by dual inhibition of PI3K 117. Rasool RU, Natesan R, Deng Q, Aras S, Lal P, Sander Effron S, signaling and Cdk9-mediated Mcl-1 transcription. Blood 2013;122: et al. CDK7 inhibition suppresses castration-resistant prostate 738–48. cancer through MED1 Inactivation. Cancer Discov 2019;9:1538–55. 137. Olson CM, Jiang B, Erb MA, Liang Y, Doctor ZM, Zhang Z, et al. 118. Sharifnia T, Wawer MJ, Chen T, Huang QY, Weir BA, Sizemore A, Pharmacological perturbation of CDK9 using selective CDK9 inhi- et al. Small-molecule targeting of brachyury transcription factor bition or degradation. Nat Chem Biol 2018;14:163–70. addiction in chordoma. Nat Med 2019;25:292–300. 138. Zhang H, Pandey S, Travers M, Sun H, Morton G, Madzo J, et al. 119. Galbraith MD, Andrysik Z, Pandey A, Hoh M, Bonner EA, Hill AA, Targeting CDK9 reactivates epigenetically silenced genes in cancer. et al. CDK8 kinase activity promotes glycolysis. Cell Rep 2017;21: Cell 2018;175:1244–58. 1495–506. 139. Iniguez AB, Stolte B, Wang EJ, Conway AS, Alexe G, Dharia NV, et al. 120. Firestein R, Shima K, Nosho K, Irahara N, Baba Y, Bojarski E, EWS/FLI confers tumor cell synthetic lethality to CDK12 inhibition et al. CDK8 expression in 470 colorectal cancers in relation to in Ewing sarcoma. Cancer Cell 2018;33:202–16. beta-catenin activation, other molecular alterations and patient 140. Bayles I, Krajewska M, Pontius WD, Saiakhova A, Morrow JJ, Bartels survival. Int J Cancer 2010;126:2863–73. C, et al. Ex vivo screen identifies CDK12 as a metastatic vulnerability 121. Morris EJ, Ji JY, Yang F, Di Stefano L, Herr A, Moon NS, et al. E2F1 in osteosarcoma. J Clin Invest 2019;129:4377–92. represses beta-catenin transcription and is antagonized by both pRB 141. Toyoshima M, Howie HL, Imakura M, Walsh RM, Annis JE, Chang and CDK8. Nature 2008;455:552–6. AN, et al. Functional genomics identifies therapeutic targets for 122. Koehler MF, Bergeron P, Blackwood EM, Bowman K, Clark KR, MYC-driven cancer. Proc Natl Acad Sci U S A 2012;109:9545–50. Firestein R, et al. Development of a potent, specific CDK8 kinase 142. Zeng M, Kwiatkowski NP, Zhang T, Nabet B, Xu M, Liang Y, et al. inhibitor which phenocopies CDK8/19 knockout cells. ACS Med Targeting MYC dependency in ovarian cancer through inhibition of Chem Lett 2016;7:223–8. CDK7 and CDK12/13. Elife 2018;7:pii:e39030. 123. Porter DC, Farmaki E, Altilia S, Schools GP, West DK, Chen M, 143. Henry KL, Kellner D, Bajrami B, Anderson JE, Beyna M, Bhisetti et al. Cyclin-dependent kinase 8 mediates chemotherapy-induced G, et al. CDK12-mediated transcriptional regulation of nonca- tumor-promoting paracrine activities. Proc Natl Acad Sci U S A nonical NF-kappaB components is essential for signaling. Sci Signal 2012;109:13799–804. 2018;11:pii:eaam8216. 124. McDermott MS, Chumanevich AA, Lim CU, Liang J, Chen M, Altilia 144. Gao Y, Zhang T, Terai H, Ficarro SB, Kwiatkowski N, Hao MF, et al. S, et al. Inhibition of CDK8 mediator kinase suppresses estrogen Overcoming resistance to the THZ series of covalent transcriptional dependent transcription and the growth of estrogen receptor posi- CDK inhibitors. Cell Chem Biol 2018;25:135–42. tive breast cancer. Oncotarget 2017;8:12558–75. 145. Ito M, Tanaka T, Toita A, Uchiyama N, Kokubo H, Morishita 125. Bergeron P, Koehler MF, Blackwood EM, Bowman K, Clark K, N, et al. Discovery of 3-benzyl-1-(trans-4-((5-cyanopyridin-2-yl) Firestein R, et al. Design and development of a series of potent amino)cyclohexyl)-1-arylurea derivatives as novel and selective and selective type II inhibitors of CDK8. ACS Med Chem Lett cyclin-dependent kinase 12 (CDK12) inhibitors. J Med Chem 2016;7:595–600. 2018;61:7710–28. 126. Clarke PA, Ortiz-Ruiz MJ, TePoele R, Adeniji-Popoola O, Box G, 146. Choi HJ, Jin S, Cho H, Won HY, An HW, Jeong GY, et al. CDK12 Court W, et al. Assessing the mechanism and therapeutic potential drives breast tumor initiation and trastuzumab resistance via WNT of modulators of the human Mediator complex-associated protein and IRS1-ErbB-PI3K signaling. EMBO Rep 2019:e48058. kinases. Elife 2016;5:e20722 147. Johannes JW, Denz CR, Su N, Wu A, Impastato AC, Mlynarski S, 127. Chao SH, Fujinaga K, Marion JE, Taube R, Sausville EA, Senderow- et al. Structure-based design of selective noncovalent CDK12 icz AM, et al. Flavopiridol inhibits P-TEFb and blocks HIV-1 replica- Inhibitors. ChemMedChem 2018;13:231–5. tion. J Biol Chem 2000;275:28345–8. 148. Quereda V, Bayle S, Vena F, Frydman SM, Monastyrskyi A, Roush 128. Lin TS, Blum KA, Fischer DB, Mitchell SM, Ruppert AS, Porcu P, WR, et al. Therapeutic targeting of CDK12/CDK13 in triple-nega- et al. Flavopiridol, fludarabine, and rituximab in mantle cell lym- tive breast cancer. Cancer Cell 2019;36:545–58. phoma and indolent B-cell lymphoproliferative disorders. J Clin 149. Rusan M, Li K, Li Y, Christensen CL, Abraham BJ, Kwiatkowski N, Oncol 2010;28:418–23. et al. Suppression of adaptive responses to targeted cancer therapy 129. Byrd JC, Lin TS, Dalton JT, Wu D, Phelps MA, Fischer B, et al. by transcriptional repression. Cancer Discov 2018;8:59–73. Flavopiridol administered using a pharmacologically derived 150. Pawar A, Gollavilli PN, Wang S, Asangani IA. Resistance to BET schedule is associated with marked clinical efficacy in refrac- inhibitor leads to alternative therapeutic vulnerabilities in castra- tory, genetically high-risk chronic lymphocytic leukemia. Blood tion-resistant prostate cancer. Cell Rep 2018;22:2236–45. 2007;109:399–404. 151. Quigley D, Alumkal JJ, Wyatt AW, Kothari V, Foye A, Lloyd P, et al. 130. Parry D, Guzi T, Shanahan F, Davis N, Prabhavalkar D, Wiswell D, Analysis of circulating cell-free DNA identifies multiclonal hetero- et al. Dinaciclib (SCH 727965), a novel and potent cyclin-dependent geneity of BRCA2 reversion mutations associated with resistance to kinase inhibitor. Mol Cancer Ther 2010;9:2344–53. PARP inhibitors. Cancer Discov 2017;7:999–1005. 131. Mita MM, Joy AA, Mita A, Sankhala K, Jou YM, Zhang D, et al. 152. Lin KK, Harrell MI, Oza AM, Oaknin A, Ray-Coquard I, Tinker AV, Randomized phase II trial of the cyclin-dependent kinase inhibitor et al. BRCA reversion mutations in circulating tumor DNA predict dinaciclib (MK-7965) versus capecitabine in patients with advanced primary and acquired resistance to the PARP inhibitor rucaparib in breast cancer. Clin Breast Cancer 2014;14:169–76. high-grade ovarian carcinoma. Cancer Discov 2019;9:210–9. 132. Hossain DMS, Javaid S, Cai M, Zhang C, Sawant A, Hinton M, et al. 153. Johnson SF, Cruz C, Greifenberg AK, Dust S, Stover DG, Chi D, Dinaciclib induces immunogenic cell death and enhances anti-PD1- et al. CDK12 inhibition reverses de novo and acquired PARP inhibi- mediated tumor suppression. J Clin Invest 2018;128:644–54. tor resistance in BRCA wild-type and mutated models of triple- 133. Bacon CW, D’Orso I. CDK9: a signaling hub for transcriptional negative breast cancer. Cell Rep 2016;17:2367–81. control. Transcription 2019;10:57–75. 154. Wyllie S, Thomas M, Patterson S, Crouch S, De Rycker M, Lowe R, 134. Bragelmann J, Dammert MA, Dietlein F, Heuckmann JM, Choidas et al. Cyclin-dependent kinase 12 is a drug target for visceral leish- A, Bohm S, et al. Systematic kinase inhibitor profiling identifies maniasis. Nature 2018;560:192–7.

march 2020 CANCER DISCOVERY | OF19

REVIEW Chou et al.

155. Sano M, Abdellatif M, Oh H, Xie M, Bagella L, Giordano A, et al. 157. Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, et al. Activation and function of cyclin T-Cdk9 (positive transcription Integrative analysis of complex cancer genomics and clinical profiles elongation factor-b) in cardiac muscle-cell hypertrophy. Nat Med using the cBioPortal. Sci Signal 2013;6:pl1. 2002;8:1310–7. 158. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. 156. Lin A, Giuliano CJ, Palladino A, John KM, Abramowicz C, Yuan ML, The cBio cancer genomics portal: an open platform for exploring et al. Off-target toxicity is a common mechanism of action of cancer multidimensional cancer genomics data. Cancer Discov 2012;2: drugs undergoing clinical trials. Sci Transl Med 2019;11:pii:eaaw8412. 401–4.

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Transcription-Associated Cyclin-Dependent Kinases as Targets and Biomarkers for Cancer Therapy

Jonathan Chou, David A. Quigley, Troy M. Robinson, et al.

Cancer Discov Published OnlineFirst February 18, 2020.

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