text:
Hana Paculová1 &
Jiří Kohoutek ORCID: orcid.org/0000-0002-4848-29561
Cell Division volume 12, Article number: 7 (2017) Cite this article
Cyclin-dependent kinases (CDKs) are key regulators of both cell cycle progression and transcription. Since dysregulation of CDKs is a frequently occurring event driving tumorigenesis, CDKs have been tested extensively as targets for cancer therapy. Cyclin-dependent kinase 12 (CDK12) is a transcription-associated kinase which participates in various cellular processes, including DNA damage response, development and cellular differentiation, as well as splicing and pre-mRNA processing. CDK12 mutations and amplification have been recently reported in different types of malignancies, including loss-of-function mutations in high-grade serous ovarian carcinomas, and that has led to assumption that CDK12 is a tumor suppressor. On the contrary, CDK12 overexpression in other tumors suggests the possibility that CDK12 has oncogenic properties, similarly to other transcription-associated kinases. In this review, we discuss current knowledge concerning the role of CDK12 in ovarian and breast tumorigenesis and the potential for chemical inhibitors of CDK12 in future cancer treatment.
Cyclin-dependent kinases (CDKs) are principal regulators of various cellular processes. They are divided into two subfamilies: cell cycle-associated CDKs (CDK1, 2, 4, 6), which directly regulate progression through individual cell cycle phases, and transcription-associated CDKs (CDK7, 8, 9, 11, 12, 13), which regulate gene transcription. These kinases phosphorylate the C-terminal domain (CTD) of Rbp1, the largest subunit of RNA polymerase II (RNA pol II) as well as various transcription regulatory factors. Since CDKs are frequently dysregulated in tumor cells, therefore they are attractive therapeutic targets for a broad spectrum of tumors [1, 2].
Eukaryotic transcription is very complex and tightly regulated. Essential cellular processes, including differentiation and response to extracellular stimuli, depend on regulation at the transcriptional level [3]. In addition, precise coordination of transcription with other events, such as mRNA processing, splicing, chromatin remodeling, and modification of histones is crucial for normal cellular physiology. Consequently, deregulation of these processes drives cancer onset and progression [4].
Transcription factors are frequently mutated in cancer cells and represent typical oncogenes and tumor suppressors. These mutations lead to alterations in the gene expression programs and might create dependency on certain transcriptional regulators making cancer cells addicted to their activities [5]. Such a phenomenon is called "Transcriptional Addiction", and it provides opportunities for novel therapeutic interventions in cancer [5].
CDK12 is a transcription-associated CDK that phosphorylates the CTD of RNA pol II and it is essential for DNA damage response (DDR), splicing, and differentiation [6]. CDK12 mutations as well as overexpression have been reported in various malignancies. Subsequently, two CDK12 inhibitors were developed independently. They will be instrumental for studying physiological function of CDK12 and could be tested as anti-cancer drugs [7, 8]. In this review, we summarize current knowledge concerning CDK12 role in tumorigenesis and its therapeutic potential.
CDKs in transcription regulation
Transcription is a complex process coordinated by numerous factors. Posttranslational modifications (particularly phosphorylation) of RNA pol II CTD constitute one of the crucial mechanisms of transcription regulation [3, 4]. RNA pol II is a multi-subunit complex responsible for RNA synthesis of eukaryotic genes coding most proteins and small RNAs. Modifications of CTD, which consists of repeated YSPTSPS heptapeptides, form patterns that enable specific binding of various factors coordinating transcription as well as co-transcriptional pre-mRNA processing [3, 4]. Phosphorylation of serines, threonine and tyrosine, among other post-translational modifications, is essential for the transition between individual phases of transcription, such as initiation, elongation, termination, and individual steps of pre-mRNA processing. The majority of kinases phosphorylating CTD of RNA pol II belong to the group of transcription-related CDKs. CDK7 is a part of transcription initiation factor TFIIH. CDK9, responsible for the activity of P-TEFb, is associated with early elongation [3, 4].
CDK12 structure, homologs and associating cyclin
In 2001, a new transcription-related kinase was discovered. It was described as a Cdc2-related kinase with an arginine/serine-rich (RS) domain (CrkRS). The CrkRS consists of 1490 amino acids and has a kinase domain, proline-rich regions and a serine-rich domain, which is typical for splicing factors from the SR protein family [9].
Later on, cyclins L1 and L2 were described as CDK12-associating cyclins and the CrkRS was renamed to CDK12 [10]. Nevertheless, the cloning of Drosophila CDK12 led to identification of cyclin K as a bona fide CDK12-associating cyclin. The cyclin K/CDK12 complex was demonstrated to phosphorylate the CTD of RNA pol II in vitro, and CDK12 was established as a CTD kinase [11]. The association between cyclin K and CDK12 was further confirmed by mass spectrometry and immunoprecipitation in mammalian cells [12, 13]. Additionally, the ability of CDK12 to phosphorylate the CTD of RNA pol II was clearly demonstrated [12]. Analogically to CDK9, CDK12 is expected to phosphorylate additional substrates other than CTD, such as transcription or splicing factors, which may be critical for CDK12 role in regulation of transcription and related processes. Additional CDK12 phosphorylation targets and related biological functions remain to be determined. The crystal structure of CDK12 was later described, opening new possibilities to study its function and its potential as a drug target and to develop specific CDK12 inhibitors [14]. The closest CDK12 human homologue is CDK13 (also known as CDC2L5, CHED). It contains a kinase domain which displays high sequence identity to CDK12 kinase domain, but its sequence is unrelated on C- and N- terminus. CDK13 associates with Cyclin K and forms a separate complex [12]. Similarly to CDK12, CDK13 is capable of phosphorylating CTD [12]. Nevertheless, CDK13 is much less studied than CDK12 and its function is less clear. Due to the sequence similarity, one can expect a redundancy or overlap in functions of these two kinases.
In yeast (S. cerevisiae), there are two kinases capable of phosphorylating Ser2 of CTD, Ctk1 and Bur1. Before CDK12 and CDK13 were discovered, it was assumed that CDK9 is the only metazoan orthologue of Ctk1 and Bur1. Based on evolutionary and functional evidence Bartkowiak et al. identified Drosophila CDK12 and human CDK12 and CDK13 as an ortholog of yeast Ctk1, while Bur1 is CDK9 orthologue [11, 15] Lsk1, less studied Ser2 CTD phosphorylating protein, is CDK12 ortholog in S. pombe [11].
CDK12 in transcription regulation
Similarly to CDK9, CDK12 is associated with transcription elongation and is able to phosphorylate the RNA pol II CTD serine at position 2 (Ser2) in Drosophila [11] and in human cells [12, 13]. However, downregulation of CDK12 activity does not affect the global transcription rate, and when CDK12 is depleted from cells, transcription of a unique subset of genes is altered (interestingly mainly the genes necessary for DDR). Depletion of CDK12 and Cyclin K results in decreased expression of long genes (> 10 kb) and genes with higher number of exons. Observations have led to the hypothesis that CDK12 is a kinase that promotes transcription of a set of specific genes [12]. Supporting this hypothesis, CDK12 was found to be necessary for the expression of Nrf2-dependent genes in Drosophila cells, where CDK12 does not affect the overall transcription but is rather involved in the transcriptional stress response [16].
Nevertheless, recent studies provide arguments against a hypothesis that CDK12 is a gene-specific CDK kinase. According to this concept, CDK12 is actively recruited to the body of transcribed genes by fully operated Pol II associated factor 1 (Paf1) right after paused RNA pol II is released into the productive elongation phase [17]. Also, when specific anti-CDK12 antibody was used in a ChIP-Seq experiment, it was found that CDK12 binds to promoters and bodies of protein-coding genes and to active transcription enhancers, with the ChIP-seq signal overlapping with the RNA pol II signal [8]. The same group developed a specific CDK12 inhibitor and identified CDK12-responsive genes in microarray experiments. Critically, a lower dose of the CDK12 inhibitor reduced transcription of core DDR genes (including BRCA1, FANCF and ERCC4) and higher inhibitor dose decreased expression of super-enhancer-associated genes compared to genes associated with typical enhancers [8].
An additional aspect related to CDK12 function in transcription is the specificity of CDK12 in relation to the specific serine within CTD. In the classical view, CDK12 phosphorylates CTD Ser2 in vivo and in vitro. In contrast to CDK9, CDK12 is responsible for Ser2 phosphorylation on the 3′ prime end of genes [8]. In an attempt to clarify CDK12 substrate preference, the Geyer group performed in vitro kinase assays and immunoprecipitation experiments. They described CDK12 ability to phosphorylate both CTD Ser2 and Ser5; however, it needs pre-phosphorylation of CTD on Ser7 for optimal activity [14]. Nonetheless, inhibition of CDK12 in HeLa cells led to a severe block of cell growth but global phosphorylation of individual CTD related serines was affected only moderately [18]. Finally, CDK12 inhibition leads to a reduction in global Ser2 but not Ser5 or Ser7 phosphorylation of the CTD [8]. To date, CTD substrate specificity studies have been dependent on antibodies against specific CTD modifications, such as H5, H14, and antibodies prepared by the Eick group [19]. The specificity of these antibodies seems to be affected by neighboring modifications [18], which could bias the results and make it challenging to draw conclusions. A cellular system suitable for mass spectroscopy analyses of CTD of RNA pol II modifications was recently developed. It provides a promising tool for elucidating the specificity of CDK12 and other CDKs in relation to individual CTD serines [20].
In addition to regulating the elongation phase of transcription, CDK12 participates in transcription termination. Polyadenylation-coupled phosphorylation of Ser2 at the 3′ end of the MYC gene by CDK12 is necessary for recruitment of polyadenylation factor CstF77 and is therefore necessary for effective transcription termination [21]. Similarly, CDK12 depletion leads to reduced Ser2 phosphorylation and cleavage stimulation factor 64 (CstF64), thereby leading to impaired 3′ end processing of the c-FOS gene after activation of EGF signaling [22]. These two studies clearly demonstrated how regulation of transcription is coupled to pre-mRNA processing by recruiting different factors to modified CTD.
It is evident that transcription elongation and termination serve as regulatory steps in gene expression. Dysregulation of these processes may alter levels of tumor suppressors or oncogenes and possibly result in tumorigenesis. Nevertheless, the exact role of CDK12 in transcription regulation is not fully understood. The fundamental question remains unanswered as to whether CDK12 affects transcription globally or if it is kinase-specific for a unique set of genes, such as DDR or super-enhancer-associated genes? Similarly to CDK9, CDK12 could bind and phosphorylate also additional factors and thus regulate transcription employing CTD phosphorylation independent mechanisms.
CDK12 function in splicing
Since its discovery, it has been known that CDK12 co–localizes with SC35 (also known as SRSF2 or SFRS2), a spliceosome component, and contains the RS domain which is typical for RNA-interacting and splicing factors [9]. Supporting CDK12 involvement in RNA-splicing machinery, three studies independently identified several factors of the splicing apparatus and components of nuclear speckles to be putative CDK12-associating partners based on mass spectrometry analyses [18, 22, 23]. Nevertheless, most of these associations have not yet been confirmed by immunoprecipitation with endogenous CDK12. One example of CDK12 involvement in splicing is based on the observation that depletion of CDK12 leads to dysregulated alternative splicing of serine/arginine splicing factor1 (SRSF1). In addition to its association with splicing factors, CDK12 was demonstrated to co-immunoprecipitate proteins of exon junction complexes and RNA-binding proteins [22]. In addition, Drosophila CDK12 is involved in the alternative splicing of neurexin IV in coordination with mRNA–binding protein HOW during Drosophila nervous system development [24].
A recent study confirmed that CDK12 is involved in splicing. The authors described interaction of CDK12 with spliceosome components and splicing regulatory factors using immunoprecipitation followed by mass spectroscopy [25]. In RNA-seq experiments, Tien et al. described a new role of CDK12 in splicing: CDK12 regulates alternative last exon splicing, gene- and cell type-specific specialized type of alternative splicing. In breast cancer cells, depletion or overexpression of CDK12 leads to altered alternative last exon splicing of a subset of genes and may contribute to tumorigenesis [25].
Despite a growing body of evidence supporting CDK12 involvement in splicing, the precise role of CDK12 in this process as well as other co-transcriptional events is yet to be elucidated. Description of additional binding factors and new potential phosphorylation substrates may clarify the precise function of CDK12 in this process. CDK12 could form a functional link between transcription regulation and co-transcriptional pre-mRNA splicing. Alternative splicing affects a large number of transcripts in mammals and provides regulation for the majority of cellular processes. Aberrant splicing of various regulatory factors also leads to tumorigenesis [26,27,28] providing one of the explanatory roles for CDK12 deficiency in tumorigenesis via splicing dysregulation.
CDK12 in development
Even though CDK12 is ubiquitously expressed, the CDK12 protein level differs in particular tissues. High human CDK12 levels can be found in testes, ovaries, leukocytes and adrenal gland, measured by mRNA levels [29]. High mouse CDK12 protein levels are in testes as well as in highly proliferative tissues and mouse embryonic stem cells [30]. This suggests that CDK12 could have tissue-specific roles in cellular commitment and differentiation. Several studies have pointed out CDK12 function in neuronal development and differentiation [31,32,33]. For instance, depletion of CDK12 (and CDK13) leads to reduced axonal outgrowth mediated probably by lowered CDK5 expression [32]. Ser2 phosphorylation in C. elegans germline depends on the activity of CDK12/cyclin K rather than on CDK9 [34].
As is evident from mouse studies, both CDK12 and its associating cyclin K are essential for early embryogenesis in mice. In vitro cultured CDK12 −/− blastocysts fail to undergo inner cell mass outgrowth due to increased apoptosis and impaired repair of DNA damage [33].
CDK12 associating cyclin K is highly expressed in murine embryonic stem cells but not in their differentiated derivatives. The cyclin K protein level decreases with differentiation and correlates with levels of Oct4, Sox and Nanog proteins known to be necessary for maintaining stemness. These observations suggest that cyclin K/CDK12 and also cyclin K/CDK13 complexes take part in maintaining the self-renewal capacity of murine embryonic stem cells [35].
One of the features of various tumors is stemness and cell dedifferentiation. Since CDK12 maintains a dedifferentiated state in mouse embryonic stem cells, one could envision a scenario where CDK12 maintains the dedifferentiated state of cancer stem cells. High CDK12 activity would therefore accelerate tumor progression and therapy resistance, paradoxically to its proposed role as a tumor suppressor.
CDK12 role in DNA damage response
Even though the exact function of CDK12 is not fully understood, it is evident that it plays a significant role in DDR by affecting the expression of genes involved in homologous recombination (HR) promoted DNA damage repair and probably also other repair pathways [7, 12, 36]. Consequently, CDK12 silencing results in increased endogenous DNA damage [12]. Cells expressing catalytically inactive mutant forms of CDK12 exhibit a compromised ability to effectively execute HR [36]. CDK12 −/− cells derived from mouse blastocysts show decreased expression of DDR genes and increased levels of DNA damage [33]. Administration of CDK12 inhibitor THZ531 also reduces expression of DDR-associated genes [8].
However, a recent study pointed out that the DNA damage pattern in ovarian tumors with CDK12 loss is different than in the case of loss of HR-associated genes. Samples bearing inactive CDK12 have been shown to contain large tandem duplications rather than markers of impaired HR [37].
Cyclin K (in addition to BRCA1) was found in a global screen for genes sensitizing cells to the DNA-damaging drug camptothecin [38]. Furthermore, downregulation of CDK12 leads not only to spontaneous cell death but also to sensitization of cells to various DNA-damaging agents such as etoposide, mitomycin C and camptothecin [12]. Also, mutant, inactive forms of CDK12 sensitize cancer cells to cisplatin [39]. CDK12 deficiency sensitizes cells to inhibitors of PARP1/2, an important factor involved in DNA repair (discussed further in more detail) [7, 39, 40].
Despite the fact that CDK12 role in DDR is not yet fully understood, it is clear that CDK12 is necessary for maintaining genomic stability and functional DDR, particularly HR promoted DNA damage repair. Impaired DDR and accumulation of DNA damage is a typical hallmark of cancer [41], which directly links CDK12 deficiency to tumorigenesis.
Transcriptional CDKs and cancer
CDKs are principal regulators of the cell cycle and consequently participate in control of cell proliferation. Each CDK plays a distinct role in this process and is activated in coordination with multiple factors. Dysregulation of CDKs is a feature typical of a large number of tumor types. Hence, CDKs are attractive targets for cancer therapy and numerous CDK inhibitors have been synthesized and tested as anti-cancer drugs [1, 42]. In addition to cell cycle-associated CDKs, transcription-associated CDKs have emerged as prospective therapeutic targets, exploiting the so-called transcriptional addiction, According to this concept, cancer cells depend on dysregulated transcriptional programs maintained by principal transcriptional regulators, among them transcription-associated CDKs [5]. An increasing number of studies have pointed out the connection between individual transcriptional CDKs and cancerogenesis [5].
CDK9, a kinase responsible for the activity of positive transcription elongation factor (P-TEFb), regulates transition from the initiation to the productive elongation phase of transcription. Overexpression of oncogenic transcription factor c-Myc leads to increased activity of CDK9 and enhances the current transcriptional program by stimulating RNA pol II elongation [43]. Consequently, CDK9 inhibitors have been shown to limit proliferation in Myc-overexpressing liver cancer cells [44] and B cell lymphoma [45]. CDK9 is activated in different types of leukemia. MLL, a histone methyltransferase, frequently fuses with components of the super-elongation complex to form oncogenic factors which activate P-TEFb and promote transcription. Inhibition of CDK9 subsequently limits proliferation of these cells [46].
After CDK13 amplification was described in hepatocellular cancer, CDK13 was consequently proposed to be an oncogene [47]. A high level of CDK11 in breast cancer correlates with clinicopathological parameters. CDK11 downregulation limits cell proliferation and migration in breast cancer cell lines. Targeting CDK11 has been proposed for breast cancer treatment [48].
In conclusion, overstimulation of transcription-associated CDKs promotes proliferation of various cancer types. Analogously, cancer cells may depend on CDK12 and thus it can serve as a therapeutic target. Importantly, CDK12 overexpression has been documented in breast tumors [49, 50].
CDK12 inhibitors
The anti-tumor potential of various CDK inhibitors has been tested in clinical trials. In addition to the pan-selective inhibitors such as flavopiridol and roscovitine, inhibitors showing specificity for individual CDKs have been developed targeting cell cycle- as well as transcription-linked CDKs [1]. Taking into account that CDK12 plays a critical role in multiple cellular processes and is mutated or overexpressed in various types of cancer, CDK12 inhibition emerges as a favorable strategy for cancer treatment. Two studies recently described CDK12 inhibitors with different chemical structure and specificity range.
Initially, dinaciclib (SCH 727965) was described as a potent inhibitor of CDK2, CDK5, CDK1 and CDK9 exhibiting an anti-proliferative effect in various cell lines [51]. Johnson et al. (2016) discovered that dinaciclib potently inhibits also CDK12 with IC50 comparable to CDK9. Dinaciclib administration, similarly to CDK12 silencing, leads to reduced expression of HR genes and reduced RNA pol II CTD Ser2 phosphorylation, and the effects of dinaciclib are thus reminiscent of CDK12 inhibition. Moreover, BRCA1 wild-type cells treated with dinaciclib exhibit compromised HR, which conveys a sensitivity to PARP1 inhibitors. Importantly, combined treatment with PARP1/2 inhibitor veliparib and CDK12 inhibitor dinaciclib efficiently inhibited tumor growth in a patient-derived xenograft model. These findings foresee a possibility to use a CDK12 inhibitor to sensitize or reverse PARP1/2 inhibitor resistance in tumors [7].
CDK7 inhibitor THZ1 has recently been shown to limit the transcription of factors dependent on super-enhancers, among them MYC proto-oncogenes, and it has been pre-clinically tested for treatment of lung carcinoma [43], T-cell acute lymphoblastic leukemia [44], and triple negative breast cancer [45]. THZ1 inhibits CDK12 at higher concentrations and its biological effect could be partly ascribed to CDK12 inhibition [52].
A selective and potent CDK12/13 inhibitor TZH531 was recently developed [8]. The attempt to synthesize a CDK12 inhibitor was based on THZ1, which covalently binds cysteine 312 located on an extension of the CDK7 kinase domain. CDK12 and CDK13 possess cysteines 1039/1017 in a similar extension close to the kinase domain. The authors exploited structural differences between CDK7 Cys312 and CDK12/13 Cys 1039/1017 and screened for an inhibitor specific solely for CDK12/13. THZ531 selectively inhibits CDK12/13 activity 50 times more efficiently than CDK7 or CDK9. In cells, THZ531 induced apoptosis, inhibited elongation of genes and led to reduced expression of DDR and super-enhancer dependent genes. THZ531 exhibited an antiproliferative effect in Jurkat T-cell acute lymphoblastic leukemia cells [8]. Yet, respective contributions of CDK12 and CDK13 to observed biological effects are not known [40].
In summary, CDK12 inhibitors show promise as anti-cancer drugs, either as a stand-alone treatment or in combination with other compounds such as PARP1/2 inhibitors.
CDK12 mutations in high-grade serous ovarian cancer (HGSOC)
Genomic instability is a typical feature of various malignancies. Mutations in DDR genes resulting in accumulation of DNA damage are often driving progressive events in cancerogenesis. Defective DNA repair machinery results in accumulation of mutations and accelerated cancer transformation and progression [41].
HR deficiency and genomic instability are characteristic for about 50% of HGSOC [53]. HR-associated genes such as BRCA1 or BRCA2 are mutated most frequently and represent typical tumor suppressors. In addition to p53, BRCA1 and BRCA2, CDK12 is one of only nine recurrently mutated genes; it is mutated in about 3% of HGSOC cases [53]. Additional studies have described these mutations in more detail. Mostly they are homozygous point mutations in the CDK12 kinase domain leading to the loss of CDK12 function [36]. Mutant CDK12 forms have compromised ability to phosphorylate RNA pol II CTD, and cells display impairment in HR promoted DNA repair. This is caused predominantly by an inability to bind cyclin K [36, 39]. Furthermore, two studies have pointed out that in patients samples, BRCA1, BRCA2 and CDK12 mutations were mutually exclusive [40, 54]. This observation strongly suggests that BRCA1 and CDK12 participate in one regulatory pathway and supports the hypothesis that CDK12 controls expression of BRCA1 and other DDR genes. This suggestion can be further supported by the fact that more of the key DDR proteins were observed to be deregulated in patient tumor samples bearing CDK12 mutations [36]. Considering these observations, CDK12 was suggested to be a tumor suppressor.
CDK12 loss confers sensitivity to PARP1/2 inhibitors
Despite its contribution to tumor promotion, genomic instability also provides an opportunity for cancer therapy. Inhibitors of PARP1, a protein which participates in DDR, require defective HR for their anti-cancer activity. As defective HR is common in some tumors, PARP1 inhibition is becoming synthetically lethal to such cells [41]. Hence, loss-of-function mutations of HR regulators BRCA1 and BRCA2 are markers of application of PARP1/2 inhibitors-based therapy [55, 56]. Although PARP1/2 inhibitors have recently been translated into clinics, certain tumors develop resistance to PARP1/2 inhibitors, and new strategies for restoring PARP1/2 inhibitors sensitivity are needed [57].
A synthetic lethality screen determined CDK12 to be one of the additional genes conferring sensitivity to the PARP1/2 inhibitor olaparib [40]. Ovarian cancer cell lines with lower expression of CDK12 are more sensitive to olaparib treatment, and downregulation of CDK12 leads to increased olaparib sensitivity. The therapeutic effect of olaparib on CDK12-silenced tumor cells was confirmed in vivo in xenograft experiments [40]. Increased sensitivity of CDK12-compromised cells to cisplatin, the alkylating agent melphalan, and the PARP1 inhibitor veliparib was observed in a CDK12-silenced ovarian cancer cell line [39]. In addition, Her2-positive breast cancer cells with downregulated CDK12 display sensitivity to PARP1/2 inhibitors [58]. Finally, the CDK12 inhibitor dinaciclib in combination with the PARP1/2 inhibitor veliparib resulted in inhibition of tumor growth in vitro, in vivo and in a patient-derived xenograft model [7].
Consistent with the fact that CDK12 is necessary for expression of HR genes, loss of CDK12 confers sensitivity to PARP1/2 inhibitors. CDK12 loss in a tumor could serve as another marker for treatment with PARP1/2 inhibitors or additional inhibitors of DDR network, as well as with other DNA-damaging compounds.
CDK12 dysregulation in breast cancer
In addition to HGSOC, several studies have shown dysregulation of CDK12 in individual subtypes of breast cancer.
Triple-negative breast cancer (TNBC) samples contain mutational spectrum similar to ovarian cancer. These tumors do not amplify any characteristic receptor (ER, HER2, or PR) but display mutations in DDR genes that promote genomic instability. Recurrently mutated genes include p53 (80% of cases) and BRCA1 (30% of cases) [59]. CDK12 mutations were identified in 1.5% of TNBC cases [59, 60]. TNBC patients with defective HR (including loss-of-function mutated CDK12) may benefit from treatment using PARP1/2 inhibitors [59].
A large number of breast tumors are dependent on an overexpressed estrogen receptor (ER) and therefore its targeted inhibition is used for counteracting the tumor growth [61]. CDK12 silencing modifies the sensitivity of ER-positive cells to tamoxifen, a drug blocking ER signaling. CDK12 downregulation activates the mitogen-activated protein kinase (MAPK) pathway, which in turn leads to loss of ER dependency and causes resistance to tamoxifen [62].
Another subtype of breast cancer is characterized by amplification of oncogene HER2 (also known as ERBB2 or EGFR2), a tyrosine kinase receptor, which stimulates cell proliferation and inhibits apoptosis [59]. In breast cancer, HER2 is a part of the frequently amplified and overexpressed 17q12-q21 locus [63]. In addition to HER2, 17q12-q21 amplicon commonly contains several neighboring genes including MED1, GRB7, MSL1, CASC3 and TOP2A [50, 59]. Interestingly, the HER2 amplicon also contains the CDK12 gene in 71% of cases [50, 64].
HER2-amplified tumors may benefit from therapy based on usage of antibodies against HER2 receptor (trastuzumab, pertuzumab) or tyrosine-kinase inhibitors (lapatinib) [59]. In addition to HER2, overexpression of co-amplified genes might also have an impact on breast cancer development. Additional genes involved in the 17q12-q21 amplicon might therefore be oncogene candidates [63]. Moreover, amplification of additional genes in the 17q12-q21 locus might be responsible for resistance of certain tumors to HER2-targeted therapy. These oncogenic factors, including CDK12, represent potential druggable targets [64]. Mertins and colleagues analyzed the proteome of breast cancer samples and found CDK12 amplification on mRNA and protein level as well as increased CDK12 phosphorylation in HER2-amplified tumors [50]. These observations indicate elevated CDK12 activity in these tumors, and CDK12 has been proposed as an additional druggable target in HER2-amplified breast tumors [50]. In parallel, the diFiore group performed a broad screen of serine/threonine kinases with altered expression in several human cancers, among them breast cancer. They found that CKD12 is upregulated in HER2–positive breast cancer samples and demonstrated a strong correlation between CDK12 level and high tumor grade. They also proposed that a high CDK12 level could serve as a prognostic marker [49].
In 13% of cases, rearrangements in 17q12-q21 amplicons lead to disruption of the CDK12 gene and resulted in CDK12 loss of function and PARP1/2 inhibitor sensitivity of these cells. This observation suggests that the subset of HER2-amplified patients with disrupted CDK12 could benefit from PARP1/2 inhibitor treatment [58]. Additionally, a fusion form of the likely nonfunctional CDK12 gene was also found in a micropapillary breast cancer sample [58].
An increasing number of studies describe loss of function or amplification of CDK12 in breast cancer samples. Loss of function in CDK12 may lead to genomic instability and be predictive of PARP1/2 inhibitor treatment. Tumors displaying CDK12 amplification, on the other hand, may be dependent on its overexpression and CDK12 may provide a new therapeutic target for breast malignancies.
Is CDK12 a tumor suppressor or does it have oncogenic properties?
Loss of tumor suppressors and addiction to oncogenes are mechanisms driving cancer onset and progression. A growing number of studies describe mutations or amplification of the CDK12 gene in tumor samples. These data may seem contradictory, since CDK12 functions either as a tumor suppressor or it has features that resemble an oncogene.
In HGSOC and TNBC, CDK12 is a tumor suppressor. CDK12 is necessary for expression of DDR genes and it is essential for HR mediated DNA repair [12, 50]. Consequently, CDK12 loss leads to increased genomic instability, which is a typical feature of these tumors and represents one of the hallmarks of cancer progression [65]. CDK12 loss-of-function mutations or inhibition confers sensitivity of cells to PARP1/2 inhibitors [7, 39, 40].
In a different context, CDK12 has properties that resemble oncogenes. CDK12 amplification, resulting in its overexpression, correlates with more aggressive tumor progression in HER2-positive breast cancers [49]. CDK12 is highly active in these tumors, and it has been proposed as a druggable target in HER2-amplified breast cancer [50]. This idea might be supported by the fact that CDK12 inhibition limits the growth of cancer cells [8, 66]. The concept of transcriptional addiction describes dependency of cancer cells on a certain transcriptional regulator, which maintains an altered transcriptional program [5]. In line with this concept, inhibitors of CDK12-related transcriptional kinases CDK7 and CDK9 reduce proliferation of cancer cells and are being tested as anti-tumor drugs. Similarly to CDK7, CDK12 inhibitors limit the expression of super-enhancer-associated oncogenic transcriptional factors [8, 67]. This suggests that certain tumors might be transcriptionally addicted to CDK12 and so CDK12 inhibition might be a promising anti-cancer strategy. A recent study described that CDK12 overexpression affects alternative last exon splicing. Therefore CDK12 overexpression can increase the invasiveness of a breast cancer cell line, by decreasing the expression of the long isoform of DNAJB6 [25].
Taken together, the dual role of CDK12 in cancerogenesis could be explained by the fact that CDK12 is essential for expression of both tumor suppressors and oncogenes, and it participates in multiple cellular processes. In different tissues and cell types, the process of tumorigenesis depends on amplification of particular oncogenes or loss of suppressors. Consequently, CDK12 can act as a tumor suppressor or its amplification can contribute to cancerogenesis depending on cellular context (Fig. 1).
Role of CDK12 in cancer. a CDK12 has the tumor-suppressive properties. CDK12 loss-of function mutations lead to decreased expression of HR genes resulting in genomic instability and tumorigenesis. CDK12 loss or inhibition sensitizes tumor cells to PARP1/2 inhibitors. b CDK12 has oncogenic properties. CDK12 amplification might lead to increased expression of various oncogenes and consequently participate in tumorigenesis. Therefore targeting CDK12 with specific inhibitors in these tumors could be beneficiary for patient treatment
CDK12 is a transcription-associated CDK essential for multiple cellular processes, including splicing, differentiation and DDR. Despite the fact that CDK12 has been extensively studied, our understanding of its functions remains limited. In vitro models will be instrumental to identify CDK12-associating factors and additional kinase targets and to elucidate whether it is a general transcriptional regulator or a specific factor for particular sets of genes. Mouse models recapitulating CDK12 loss or gain of function will be illustrative in studying particular aspects of diseases and development. Elucidation of CDK12 functions would lead to better assessment of its roles during tumorigenesis.
CDK12 mutations and amplification in tumors have been documented in an increasing number of studies. Recently developed CDK12 inhibitors constitute not only powerful research tools but also promising anti-cancer drugs. CDK12 inhibitor monotherapy could be useful for cancer patients tumors with overexpressed and activated CDK12. CDK12 inhibition increases sensitivity of cells to PARP1/2 inhibitors, thus presenting a potential strategy for targeting PARP1/2-resistant tumors.
cdk:
cyclin-dependent kinase
C-terminal domain
RNA pol II:
DDR:
DNA damage response
CrkRS:
Cdc2-related kinase with an arginine/serine-rich (RS) domain
P-TEFb:
positive transcription elongation factor b
Paf1:
pol II associated factor 1
Nrf:
nuclear respiratory factor
CstF77:
cleavage stimulation factor 77 kDa subunit
cleavage stimulating 34 factor 64
SC35:
SR splicing factor 2
SFRS1:
serine/arginine splicing factor1
c-MYC:
MYC proto-oncogene
HGSOC:
high-grade serous ovarian cancer
PARP:
poly (ADP-ribose) polymerase
ER:
estrogen receptor
HER-2:
human epidermal growth factor receptor 2
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HP and JK wrote the manuscript. Both authors read and approved the final manuscript.
The authors would like to thank prof. BM Peterlin, Dr. Sabina Sevcikova and members of the J. Kohoutek laboratory for critical discussion and their suggestions during the preparation of this review.
The authors certify that they have no affiliations with, or involvement in, any organization or entity with any financial or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.
The authors agree with publishing this manuscript.
This study was supported by the Ministry of Health of the Czech Republic, Grant 16-34152A and by the Ministry of Agriculture, Grant RO0517 to J.K.
Department of Chemistry and Toxicology, Veterinary Research Institute, Hudcova 296/70, Brno, 621 00, Czech Republic
Hana Paculová & Jiří Kohoutek
Hana Paculová
Jiří Kohoutek
Correspondence to Jiří Kohoutek.
Paculová, H., Kohoutek, J. The emerging roles of CDK12 in tumorigenesis. Cell Div 12, 7 (2017). https://doi.org/10.1186/s13008-017-0033-x
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Yahoo Life UK
As Princess Eugenie announces pregnancy, who are the late Queen's other grandchildren and great-grandchildren?
·Royal Correspondent
24 January 2023, 12:01 pm ·20-min read
The Queen with her grandsons Prince William and Prince Harry. (Getty Images)
Queen Elizabeth II may well have been the world's most famous grandmother. Boasting eight grandchildren and 12 great-grandchildren at the time of her death, she was the proud matriarch of a huge Royal Family.
Now, the late monarch's family is expanding further, as Princess Eugenie announced she is expecting her second child with husband Jack Brooksbank.
Who are the late Queen Elizabeth II's grandchildren?
In 1977, the late Queen welcomed her first grandchild when her daughter Princess Anne had her son, Peter Phillips, and the latest addition to the family will be due in the summer, and the first since Her Majesty's passing in September 2022.
Princess Eugenie's first child, August, was born without a title, despite his mother being a princess. However, it has been common for the Queen's grandchildren and great-grandchildren to take varying titles, or be brought up with none at all.
With a mixture of untitled commoners and heirs to the throne, here are the Queen's grandchildren and great-grandchildren.
Peter Phillips (born 15 November, 1977)
Queen Elizabeth II with her daughter, Princess Anne and grandson, Peter Phillips at the baby boy's christening, in December 1977. (Getty Images)
Peter is the eldest and only son of Anne, the Princess Royal, and her former husband Captain Mark Phillips.
He born in November 1977, with his birth marked by a 41-gun salute. He was baptised in the Buckingham Palace music room a week later, but his mother opted not to give him a royal title.
The lack of title means he is simply "Mr Peter Phillips" and he is not known as His Royal Highness. He was fifth in line to the throne when he was born.
Phillips went to Gordonstoun in Scotland, like his late grandfather Prince Philip, and even became head boy. After school, he went to the University of Exeter where he completed a degree in sports science.
His previous relationships include cod liver oil heiress Elizabeth Lorio and flight attendant Tara Swain.
But it was love at first sight when he met Autumn Kelly in Canada in 2003. The couple were together for about four years before he proposed in 2007, and married in Windsor a year later.
Peter and Autumn Phillips at Cheltenham Racecourse in 2020. Despite separating, the pair have remained close. (Getty Images)
They have two children, Savannah and Isla.
But in February 2020, the Peter and Autumn confirmed they had split up and said in a statement that they had told their families at the end of 2019.
He doesn't carry out any official royal duties but is of course still invited to Royal Family events like Trooping the Colour.
Zara Tindall (born 15 May, 1981)
Princess Anne leaving the hospital with Zara in 1981. (Getty Images)
Zara Tindall is the only daughter of Princess Anne and Captain Phillips, and the younger sister of Peter.
She was born in May 1981, and baptised at Windsor Castle. She was also never given a title, meaning she is not an HRH.
Like her brother and her late grandfather, she attended Gordonstoun, and also followed in Phillips' footsteps to go to Exeter.
Tindall is an accomplished equestrian, competing at the London Olympics in 2012, and even winning BBC Sports Personality of the Year in 2010.
Zara and Mike Tindall attending Wimbledon 2022. (Photo by Karwai Tang/WireImage)
Read more: Zara and Mike Tindall's relationship in pictures
Tindall is married to former England rugby player Mike Tindall, and the couple have three children, Mia, Lena and Lucas.
She's previously spoken about how two of her pregnancies ended in miscarriage.
Prince William (born 21 June, 1982)
Diana holding Prince William at his christening in 1982. (Getty Images)
First in line to the throne, Prince William, Prince of Wales is the eldest son of King Charles and the late Diana, Princess of Wales.
William was born in June 1982 and baptised in the music room at Buckingham Palace in September of the same year.
Prince Charles was keen not to send his children to Gordonstoun, having not enjoyed his time there (he once called it "Colditz in kilts"), so William was sent to Eton College in Windsor.
But William did venture to Scotland for university, studying at St Andrews after a gap year, where he met his now wife, Kate Middleton.
Prince William is first in line to the throne. (Photo by Max Mumby/Indigo/Getty Images)
The Prince and Princess of Wales dated for about seven years – though split briefly in 2006 – and confirmed their engagement in November 2011.
The couple now have three children of his own – Prince George, Princess Charlotte and Prince Louis.
Prince Harry (born 15 September, 1984)
Prince Harry with Diana on the day of his christening. (PA Images)
William's younger brother, Prince Harry is the second son of Charles and Diana. He was born in September 1984.
His name is actually Henry, and he was baptised as such in St George's Chapel in Windsor.
The Duke of Sussex also went to Eton College, and was a house captain of games, showing his love for all things sport. He took a gap year before starting his military career at Sandhurst.
Harry served for 10 years in the army, including a tour in Afghanistan.
Harry has had several girlfriends over the years and been linked with other women. He dated Chelsy Davy, but their relationship broke down because of media attention. He was also with Cressida Bonas for a number of years before they split.
He met the actress Meghan Markle in early 2016 and their relationship was secret for several months. They announced their engagement in November 2017.
Harry and Meghan ahead of the Queen's funeral in September 2022. (Photo Danny Lawson - WPA Pool/Getty Images)
Harry and Meghan married at St George's Chapel, Windsor in May 2018. They have one son, Archie Harrison and a daughter, Lilibet Diana.
In January 2020, they announced they wanted to step back as senior members of the Royal Family and intended to carve their own path which they hoped would allow them to represent the Queen and earn their own money.
However the Royal Family was not so keen to allow the middle ground and the couple had to agree not to use the HRH title and to stop receiving money from the crown estate.
They moved to California and now own a home in Santa Barbara. Meghan has her own Spotify podcast, named Archetypes, and the couple are working on a Netflix documentary.
In July 2020 the duchess revealed she had suffered a miscarriage.
Princess Beatrice (born 8 August, 1988)
Sarah Ferguson and Prince Andrew at Princess Beatrice's Christening in 1988. (Getty Images)
The oldest daughter of Andrew, the Duke of York, and Sarah Ferguson, Beatrice was born in August 1988.
Beatrice went to Coworth Park School and then to St George's in Ascot, where she was head girl.
Although she has a royal title, Princess Beatrice is not a working royal, and earns her own money working in business. She is a patron of several charities and organisations.
Princess Beatrice announced her engagement to the property developer Edoardo Mapelli Mozzi in September 2019.
Beatrice visiting her wedding dress on display in Windsor Castle after her secret wedding. (Getty Images)
They had planned a wedding in London for May 2020, complete with a grand reception in Buckingham Palace, but it was scuppered by the coronavirus pandemic.
Instead they married in secret in July 2020, with Beatrice borrowing a dress from the Queen for the small occasion.
Princess Eugenie (born 23 March, 1990)
Sarah, Duchess of York leaves the Portland Hospital in London with her new daughter, Princess Eugenie, in March 1990. (Getty Images)
Eugenie is the second daughter of Andrew and Sarah and was born in March 1990.
As a child, she suffered scoliosis and had to undergo surgery to correct the curvature of the spine. She had titanium rods and screws inserted into her spine, and was in wheelchair for about six days thereafter.
Eugenie also went to Coworth Park School, and then to Marlborough College in Wiltshire. She read her degree at Newcastle University.
Like her sister, she works full time, and attends events as a member of the Royal Family but not as a working royal. She works in the art world.
Jack Brooksbank and Princess Eugenie at St George's Chapel after their wedding ceremony in October 2018. (Getty Images)
Princess Eugenie married Jack Brooksbank in October 2018 at St George's Chapel in Windsor, and chose a dress to show her scoliosis scar.
The couple welcomed their first child, August, in February 2021, and announced they were expecting their second child in January 2023.
The baby will be the Queen's 13th great-grandchild.
Lady Louise Windsor (born 8 November, 2003)
The Earl of Wessex holding Lady Louise Windsor as they leave hospital in Frimley. (Getty Images)
Louise is the Queen's youngest granddaughter and the firstborn child of Edward, the Earl of Wessex, and his wife Sophie.
She is known as Lady Louise Windsor. She was born prematurely in November 2003, while her father was away, so he was not present at her birth.
She was baptised in April 2004 in the private chapel at Windsor Castle. She was the last royal baby to wear the original christening gown.
Louise was born with an eye condition which causes one eye to turn inwards. She had an operation to correct it, which was unsuccessful, but more treatment in 2013 restored her sight.
Lady Louise Windsor and Sophie, Countess of Wessex visit The Wild Place Project at Bristol Zoo in 2019. (Getty Images)
She is largely out of the public eye at the moment, but was a bridesmaid at William and Kate's 2011 wedding, and is also seen at events like Trooping the Colour.
Lady Louise has also taken up carriage driving, something her late grandfather did.
James, Viscount Severn (born 17 December, 2007)
Sophie and Edward welcomed baby James in 2007. (Getty Images)
The Queen's youngest grandchild, James, was born in December 2007.
Like his sister, he has a courtesy title, and so is known as the Viscount Severn. His parents probably chose this to avoid some of the pressure of a royal title.
He is 12th in line to the throne and was also baptised in the private chapel at Windsor Castle. He was the first to wear the new replica christening gown.
Both he and his older sister have lakes named after them in Canada, done to mark his father's visit to Manitoba.
James, Viscount Severn watches the swimming at the 2022 Commonwealth Games. (Photo by Max Mumby/Indigo/Getty Images)
His title reflects his mother's Welsh roots, as the River Severn rises in Wales.
James and Louise travelled with their parents on royal tour to South Africa in 2015.
Who are the late Queen Elizabeth II's great-grandchildren?
Savannah Phillips (born 29 December, 2010)
Savannah Phillips with Prince George and Princess Charlotte at the Platinum Jubilee. (Photo by Max Mumby/Indigo/Getty Images)
Savannah Phillips is the oldest daughter of Peter Phillips and his former wife Autumn, and the Queen's first great-grandchild.
She was born in 2010, at Gloucestershire Royal Hospital and was christened the next year at the Church of the Holy Cross in Avening.
Savannah's middle names are Anne Kathleen. Anne is a nod to her grandmother, Princess Anne.
Savannah lives on the Gatcombe Park Estate. After her parents announced their separation, they said they would continue to live nearby, but the exact nature of this arrangement isn't known.
Savannah Phillips and Prince George on the balcony of Buckingham Palace during Trooping The Colour in 2018. (Getty Images)
Despite not having a royal title, and the fact she will never carry out royal duties, Savannah is placed in the line of succession.
She does attend Royal Family events like weddings and Trooping the Colour, and delighted fans by making mischief with her cousin Prince George on the balcony of Buckingham Palace.
Isla Phillips (born 29 March, 2012)
Isla Phillips attends a Service of Thanksgiving for the life of Prince Philip at Westminster Abbey in March 2022. (Photo by Max Mumby/Indigo/Getty Images)
Isla Phillips is the second daughter of Peter and Autumn Phillips and the younger sister of Savannah.
She was born in March 2012. She also has a place in the line of succession, but no royal title.
Her middle name is Elizabeth, after her great-grandmother, the Queen.
While she was born at the same hospital as her older sister, she was christened at a different church, St Nicholas Church in Cherington.
Neither of the girls' godparents were officially announced, likely because of reduced interest and coverage over them as they are further away from the throne and their father is not a senior royal.
Isla Phillips, wearing animal design face paint, at the 2019 Festival of British Eventing at Gatcombe Park. (Getty Images)
Isla was a bridesmaid at the wedding of Princess Eugenie and Jack Brooksbank at Windsor Castle in 2018.
Her childhood has been relatively private, though she is often photographed at events held by her parents and grandparents at the Gatcombe Park estate, like the Festival of Eventing.
Mia Tindall (born 17 January, 2014)
Zara with her daughter Mia in 2014 at a Horse Trials event. (Getty Images)
Mia Tindall is the oldest daugher of Zara and Mike Tindall. She was born in January 2014, and her middle name is Grace.
Her father announced the name of his daughter on Twitter.
Mia lives near to her cousins, the Phillips, as the Tindalls also have a home on the Gatcome Park Estate.
Like older cousins Savannah and Isla, Mia was born in the Royal Gloucestershire Hospital. She was christened at the St Nicholas Church in Cherington.
Mia was homeschooled by her parents during the coronavirus pandemic, but her dad joked about being keen to get her back to her lessons in school as soon as possible.
Mia Tindall eats an ice cream at the Whatley Manor Gatcombe International Horse Trials at Gatcombe Park in 2019. (Getty Images)
He has also shared some behind the scenes of their lessons, including his brightly painted face when Mia was let loose.
Mia is seen at general Royal Family events like Trooping the Colour and the events on Gatcombe Park estate.
Lena Tindall (born June, 2018)
Mike Tindall carries daughter Lena on his shoulders at the 2019 Festival of British Eventing at Gatcombe Park. (Getty Images)
Lena Tindall is the second daughter of Zara and Mike Tindall. She was born in 2018, after the couple had suffered a miscarriage.
Lena is now a big sister and middle child as Zara had her third child in March 2021.
Lena was born in Stroud Maternity Hospital and shares a middle name with her cousin, as hers is also Elizabeth.
She was christened at St Nicholas Church in Gloucestershire and it has previously been reported that Prince Harry is her godfather.
She also does not have a royal rank and is highly unlikely to ever be a working royal as her mother is not.
Lucas Philip Tindall (born March, 2021
Mike Tindall with his son Lucas at the Festival of British Eventing in August 2022. (Getty Images)
On 21 March 2021, the third Tindall was born.
Lucas Philip Tindall was born on the bathroom floor of the family home in Gloucestershire, following a night of contractions for mum Zara.
Lucas weighed 8lbs 4oz. He is the first son for the Tindalls, and the first grandson for Princess Anne.
Prince George of Cambridge (born 22 July, 2013)
Second in line to the throne, Prince George is the oldest son of the Prince and Princess of Wales.
George was born in July 2013 in the Lindo Wing of St Mary's Hospital in Paddington, London.
The Duke and Duchess of Cambridge leave the Lindo Wing of St Mary's Hospital in London, with their newborn son, Prince George of Cambridge. (PA Images)
George was christened George Alexander Louis at the Chapel Royal in St James's Palace by Archbishop of Canterbury Justin Welby in 2013.
He attends Lambrook School in Berkshire, having previously been at Thomas's Battersea, a private school in south London.
He was homeschooled throughout much of the coronavirus pandemic, at the family country home of Anmer Hall, Norfolk.
His parents have been keen to give him a normal upbringing, despite the fact that one day he will be king.
He has accompanied his parents on a number of their tours abroad, including trips to Australia, Canada, Poland and New Zealand.
However he has mostly had to stay at home while his parents worked since he started school.
Prince George, Prince Louis and Princess Charlotte with the Prince and Princess of Wales. (Getty Images)
Photographs of George are released each year to mark his birthday, and he attends some events with his parents.
In December 2020, royal fans were treated to pictures of him with his younger siblings and parents at a night out at the theatre to thank key workers.
Princess Charlotte of Cambridge (born 2 May, 2015)
Princess Charlotte of Cambridge being held by her mother outside the Lindo Wing. (Zak Hussein/Corbis)
Princess Charlotte is the only daughter of the Duke and Duchess of Cambridge and Cornwall.
She was born in May 2015 in the Lindo Wing of St Mary's Hospital, and christened at the Church of St Mary Magdalene, in Sandringham.
She was given the full name Charlotte Elizabeth Diana – a nod to her grandfather, Prince Charles, her great-grandmother, the Queen, and her late grandmother, Princess Diana.
Charlotte followed in her brother's footsteps, and attends Lambrook School too.
During the lockdown, she was taught at home with her brother by her parents. Kate joked that her lessons were more exciting than her older brothers, and revealed she was reading 'Spider Sandwiches'.
Princess Charlotte attends the Christmas Day Church service at Church of St Mary Magdalene on the Sandringham estate in 2019. (Samir Hussein/WireImage)
Charlotte has also been on some royal tours with her parents and brother, including to Canada. She enjoys waving from the Buckingham Palace balcony, as she has been able to during Trooping the Colour.
In 2019, she and her brother attended the Christmas Day service at St Mary Magdalene in Sandringham for the first time, though they had been pictured attending church on Christmas Day previously.
Prince Louis of Cambridge (born 23 April, 2018)
Prince Louis in 2018, leaving the Lindo Wing with his parents. (Getty Images)
Prince Louis was born in April 2018, the third child of William and Kate. He is fifth in line to the throne.
He is the first brother of an older sister not to displace her in the line of succession. Thanks to a change in the law which came into effect in 2013, Charlotte retains her place as fourth in line despite Louis being a boy.
It will mean her children will be above him and his children as well.
He was also born in the Lindo Wing and was christened in the Chapel Royal at St James's Palace.
Louis also attends Lambrook School. He has made public appearances on the balcony at Buckingham Palace and at the theatre with his family in December 2020.
Prince Louis on his first day at Lambrook School in September 2022. (Photo by Jonathan Brady - Pool/Getty Images)
However he has not been on a royal tour.
Louis also appeared in a number of videos during the lockdown including one in which he and his siblings asked questions to Sir David Attenborough and another where they clapped for carers.
Archie Harrison Mountbatten-Windsor (born 6 May, 2019)
Harry and Meghan held a photocall for their son two days after he was born. (Getty Images)
Archie Harrison Mountbatten-Windsor was born in May 2019, the son of Prince Harry and Meghan Markle.
Archie is seventh in line to the throne, but his parents declined the offer of a royal title on his birth, instead choosing to give him the family surname of Mountbatten-Windsor.
The young royal has been brought up very different to his cousins, the Cambridges. Harry and Meghan waited until two days after his birth to hold a photocall for the watching world to get a glimpse of him, and his christening was held in private.
The names of his godparents were not officially released.
Since his parents opted to step back from their senior royal roles, he has been mostly brought up in Canada and the US, where he now lives in California. The couple have sought to keep many details of his life private, and have not released many photos of him.
A stylised image was released over Christmas of the young royal with his parents in their California garden, and he was heard for the first time on a recording around the same time as he made a brief appearance on the first Archewell Audio podcast.
Lilibet 'Lili' Diana Mountbatten-Windsor (born 4 June, 2021)
Lilibet Diana Mountbatten-Windsor, the daughter of the Duke and Duchess of Sussex, was born in 2021.
Harry and Meghan announced the birth of their second child in June 2021.
Lilibet, also known as Lili, was born on 4 June in Santa Barbara Cottage Hospital in Santa Barbara, California.
A statement from the couple's spokesman said: "Lili is named after her great-grandmother, Her Majesty The Queen, whose family nickname is Lilibet.
"Her middle name, Diana, was chosen to honour her beloved late grandmother, The Princess of Wales.
"This is the second child for the couple, who also have a two-year-old son named Archie Harrison Mountbatten-Windsor. The Duke and Duchess thank you for your warm wishes and prayers as they enjoy this special time as a family."
They shared a photo of Lili celebrating her first birthday at Frogmore Cottage in Windsor.
August Philip Hawke Brooksbank (born 21 February, 2021)
Jack Brooksbank with his parents at the Platinum Jubilee in 2022. (Photo by Mark Cuthbert/UK Press via Getty Images)
August Brooksbank is the first son of Princess Eugenie and Jack Brooksbank was the Queen's youngest great-grandson for a month before Zara had her third child.
He was born at 8.55am on 9 February 2021 at the Portland Hospital in London, the same hospital where Eugenie was born.
He weighed 8lbs 1oz.
His full name is August Philip Hawke. He is named after his great-grandfather and his two great x5 grandfathers.
Sienna Elizabeth Mapelli Mozzi (born 19 September, 2021)
2021 was a busy year for royal babies, with the Queen's youngest great-grandchild, Sienna Elizabeth Mapelli Mozzi, being born after August and Lucas in the same year.
Princess Beatrice and her husband Edoardo Mapelli Mozzi welcomed their first child on 18 September 2021.
Sienna Elizabeth was born at Chelsea and Westminster Hospital, London, and stands 10th in line to the throne.
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Toyota to replace Akio Toyoda as president and CEO
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India's equity Mutual Funds to move to two-day settlement
Equity mutual funds in India will be settled within two days starting February, instead of the prevalent three day settlement cycle, an industry body said on Friday. The move for a shorter settlement period for equity mutual funds follows Indian markets completing a shift to a one-day settlement cycle on Friday. "It has been decided all Asset Management Companies (AMCs) will move to T+2 redemption payment cycle for equity schemes, and implement this uniformly with effect from Feb. 1, 2023," the Association of Mutual Funds in India said in a statement.
Adani Group firms lose $9bn in value amid short-seller claims
Hindenburg Research says world's third-richest person, Gautam Adani, is pulling 'largest con in corporate history' Adani Corporate House in Ahmedabad, India. Photograph: Ashish Vaishnav/SOPA Images/REX/Shutterstock
Britain opposed to Russia, Belarus athletes being at Paris Olympics
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Tata Motors to raise prices of passenger cars by 1.2%
BENGALURU (Reuters) -Indian carmaker Tata Motors Ltd said on Friday it would hike prices of its passenger vehicles by 1.2% on a weighted average basis from Feb. 1, citing a rise in overall input costs. The price increase comes a month after the maker of the Tiago and Harrier cars raised commercial vehicle prices by up to 2% in January.. Earlier this month, rival and India's top car maker Maruti Suzuki raised prices by an average of 1.1% across its models, blaming similar cost pressures.
Jailed Kurdish leader urges unity against Erdogan
Turkey's pro-Kurdish party should back the main opposition candidate instead of fielding its own against President Recep Tayyip Erdogan in May elections, its elder statesman told AFP from jail."I am in favour of backing a joint candidate," Selahattin Demirtas, who ran against Erdogan twice, told AFP through a lawyer from his jail in the western city of Edirne.

redpajama_set_name: RedPajamaCommonCrawl


After the first year, continuing matriculated graduate students are given priority for housing for a specified number of years based on their academic degree program. Master's students are given one additional year of limited priority for housing. Doctoral students are given five additional years of limited priority for housing. Limited priority years are not automatically cumulative, so students do not receive additional years of limited priority for subsequent degrees. If a student completes a master's program and then moves to a doctoral program, they receive four additional limited priority years, which is the difference between the allocation for a master's and a doctoral program. Students who live in residences that are open year-round and who remain in continuous occupancy in their rooms or apartments may renew their contracts annually if they meet certain eligibility requirements. Students who live in residences that are open only during the academic year, or who want to change residences, re-enter the lottery each year. Approximately 80% of continuing student applicants are assigned housing each year.


text:
Mount & Blade: Warband GOG.COM Key GLOBAL
Mount & Blade: Warband is a standalone expansion to Mount & Blade – an action role-playing game developed by TaleWorlds Entertainment and published by Paradox interactive in 2010. The game is an unique action RPG game, i ...
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Mount & Blade: Warband is a standalone expansion to Mount & Blade – an action role-playing game developed by TaleWorlds Entertainment and published by Paradox interactive in 2010. The game is an unique action RPG game, in which players take the role of a knight and guide their character through a long journey full of adventures during which they can become a general guiding his forces in one of the battles for the supremacy on the continent, gaining fame, fortune and most of all, power.
What's new in Mount & Blade: Warband?
Mount and Blade: Warband offers a completely new faction the Sarranid Sultanate, inhabiting the desert areas of Calradia. The Sarranids are a versatile faction, very useful in skirmishes, with high Athletic skill. They are adept at using polearms and thrown weapons and possess a very powerful cavalry units.The new gameplay mechanics introduced in Warband include the bandit camps, that appear in various locations and often serve as the objectives of quests for local lords.
Bandit camps can be raided, and victory against them results in the destruction of the camp and gaining loot, such as weapons and armor, as well as supplies for the villages under players' protection. However, if the player loses the skirmish, the bandits will move their camp somewhere else and will continue to terrorize the area. The bandit camps also respawn if the player wins the battle.
Other major mechanic changes include:
Automated blocking, which will guard the player character from incoming missiles, even if his shield is lowered,
Chamber blocking, which enables parrying and immediately attacking the enemy,
Couched lance mechanic, where players can deal significant damage from horseback when armed with a lance-type weapon,
Founding and commanding players' own kingdom,
Differentiated ransom system (the amount of ransom depends on the class of the prisoner),
Balancing the enemy party numbers to match the players',
Buying land to set up goods production enterprises
Marrying other vassals
Warband introduces multiplayer mode to the series, where up to 64 players can compete against each other. Player characters can be customized with money received for killing other players.
Mount & Blade: Warband is an action RPG game, which emphasizes open world gameplay style. The player is thrown into the land of Calradia, where they can join factions, create their own kingdoms, become a mercenary or even take no side in the ongoing conflict and become an outlaw. The game has no storyline and leaves the player free choice of how to proceed in the world of Mount & Blade. Before the player can start their adventure, they will have to customize their character. He will have to decide on his character's gender and past which influences their starting attributes. The player will then proceed to create the character's appearance.
The game world map can be traveled through by clicking on the desired destination. Should the player encounter a conflict along the way, they can decide to avoid it or engage the enemy party. Players can fight either on horseback or on foot. Dealt damage depends on several factors such as weapon type, skill, quality of the weapon, and character's speed compared to that of the enemies as weapons used while running or riding can deal bigger damage. Another important aspect of the combat is the effective minimal and maximal weapon range, for example, a spear will deal more damage from a distance than a hammer and vice versa.
Winning the battle results in increased weapon proficiency as well as being awarded a Renown, which increases the players' standing in the game world. High Renown might gain attention from the lords, who will offer the player vassalage. As a vassal, players are given control of a small fief, from which they can collect taxes.
Battles can be waged in open field, tournament arenas, during siege scenarios in which the player is tasked with either attacking or defending a fortification, or in settlements after triggering a specific scenario like bandit raids, being caught on sneaking into enemy territory or an ambush.The player can form a party which will travel with him around the game world and together engages in combat. The number of party members is decided by the level of the player's "Leadership" skill and renown.
Both players and critics praised Warband for its enjoyable gameplay, several improvements over the original title and engaging multiplayer. Most gaming journalists and players praised better graphics and new multiplayer modes that make Mount and Blade: Warband a must-have for not only the fans of the original game but for fans of RPG and strategy games overall.
If you're looking for quintessential medieval game experience, with storming castles, warding off bandits, and fighting in tournaments, Mount & Blade: Warband for PC is a game you should have in your collection.
Robust open world
Faithful representation of the medieval lifestyle
Engaging class-based combat
Multiplayer mode hosting up to 64 participants
An interesting hybrid of RPG and a Real-Time Strategy
A stand-alone expansion pack to the popular Mount&Blade game
Graphical overhaul: Support added for HDR, FSAA, depth of field, soft particles, tone mapping, and many other effects
New models with greater detail and high-quality textures,
Multiplayer battles with up to 64 players. Multiplayer modes include Deathmatch, Team Deathmatch, Capture the Flag, Conquest, Battle, and Siege, A campaign allowing you to become the ruler of a faction and convince lords to become your vassals,
The ability to upgrade your companions to vassals by granting them lands, The ability to marry a lady of the realm for romance or cold political gain. Try to win a lady's heart through poetry or bravery,
Improved mechanics for soldier morale: Soldiers will break and run away if their morale gets too low, Pick any projectile off the battlefield for use as additional munitions New motion-captured combat animations,
Numerous improvements to the combat system: Your shield will still stop arrows even if you are not actively defending, The ability to play multiplayer matches on random maps as well as hand-designed ones,
Multiplayer equipment system: Earn money by fighting opponents or accomplishing goals,
The ability to use most throwing weapons in close combat: Switch to using a javelin as a short spear when the enemy gets close,
Spend gold on more powerful equipment, using a carefully balanced system that will make combat more exciting without giving too much of an advantage to the leading team.
Below are the minimum and recommended system specifications for Mount & Blade: Warband GOG.COM Key GLOBAL. Due to potential programming changes, the minimum system requirements for Mount & Blade: Warband GOG.COM Key GLOBAL may change over time.
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NWSL Season Preview: Thorns hungry for more in defense of title
Allison Lee
2017: 14-5-5 (2nd place in regular season, NWSL Champions)
Head Coach: Mark Parsons (3rd season with Thorns)
Home Ground: Providence Park
'17 in Review: Last year the Portland Thorns entered the 2017 season as the NWSL Shield holders and, as a result, were a favorite to win it all. However, the legitimacy of that seemed in question when midfielder Tobin Heath was sidelined for the vast majority of the season with a lingering back injury. It was uncertain how the team would survive without one of their star players, but a shift in positions for Christine Sinclair helped fill the void left by Heath's absence. Sinclair's attacking prowess, combined with another stellar season for Nadia Nadim and the best season in Hayley Raso's NWSL career, helped Portland stay ahead of their opponents offensively.
On the other side of the field, goalkeeper Adrianna Franch grew over the course of the season into the best goalkeeper in the league, thanks partly to the tutelage of goalkeeper coach Nadine Angerer. Franch's improved decision making, steady hands, and solid distribution made her a imposing presence in goal for the Thorns. She was helped by one of the best defenses in the league being in front of her, and together the Thorns' back five led the league in shutouts.
Their biggest shutout of the season came in the biggest game of the year, the NWSL Championship. Although the Thorns finished second in points behind the North Carolina Courage in the regular season, they did what they had been unable to do in 2016 and advanced to the NWSL final, where they defeated the Courage, 1-0, to win their second league title.
What's New: Although the Thorns experienced one of the less tumultuous offseasons in the league, it was also not the easiest for Portland fans, as they had to say goodbye to some of the players they had grown to love watching play at Providence Park.
Two of the Thorns' most commanding players, Amandine Henry and Nadia Nadim, returned to Europe after the final, signing with Olympique Lyonnais and Manchester City Women, respectively. The Thorns will also be without Icelandic midfielder Dagny Brynjarsdottir throughout the 2018 season, as she is expecting her first child.
USWNT midfielder and Portland Thorns original Allie Long was sent to the Seattle Reign in a trade that included the rights to Aussie Caitlin Foord, and former University of Florida standout Savannah Jordan, who joined the Thorns partway through her rookie season after a short stint in Scotland, was traded to Houston.
Kendall Johnson, who saw little playing time last season as she continued recovering from injury, was waived by the Thorns shortly after the NWSL Championship and has spent the 2018 preseason in camp with the Utah Royals.
Despite the losses, Portland fans had reason to celebrate, as the players Parsons brought in to fill in the roster vacancies would be key additions to any roster in the league. As previously mentioned, the Thorns acquired the rights to former Sky Blue FC defender Caitlin Foord in the trade for Long, and after a two-year leave of absence from the NWSL, the versatile Aussie's return to the league is imminent.
The Thorns also signed another member of the Matildas this offseason, defender Ellie Carpenter. Although Carpenter won't join the Thorns until after her 18th birthday (April 28), Parsons is high on the teen defender, saying she has the potential to be "world class" and an asset to the Thorns.
However, the player Mark Parsons seemed most keen on joining the Thorns is Brazilian midfielder Andressinha, who was acquired from the Houston Dash in the Jordan trade. Although Andressinha's arrival to the team has been delayed by national team duty and a knock picked up during it, Parsons is excited about what she'll bring to the team.
"I'm excited for her to join us because players like her is the reason why I fell in love with this sport and this game. She has a wonderful brain, great technique, and she sees things and feels things that not many players in the world do feel and see," he said. "I'm excited for our team for her and for my soul that I get to watch this type of player day in and day out."
Margaret Purce, Ifeoma Onumonu, and Angela Salem also joined the Thorns this year by way of the dispersal draft, and Purce and Onumonu put in solid performances during the Thorns Spring Invitational preseason tournament.
The Thorns also had another solid draft class, taking midfielders Sandra Yu and Gabby Seiler and goalkeeper Annabella Geist with their three picks.
If things click: Any team coming off an NWSL Championship win will be an early favorite to return to the playoffs, especially with a roster like Portland's, but so far only one team in the league has ever won back-to-back titles: the now-defunct FC Kansas City.
For Parsons, as much as the championship win was an "amazing victory," the Thorns not only want to repeat as league champions, they want to be even better than before.
"Last year…we didn't fulfill everything that we set out to achieve. We didn't win the NWSL Shield and the Championship," he said. "We didn't play our best until the second half of the season. I think we finished, I think it was 11-1-1, the record the last 13 games, including the playoffs. And I might be twisting that, but the record is unbelievable. But it took us half a year to get to that, and that's leaving us unfulfilled."
Getting off to a better start won't be easy for the Thorns though, which Parsons is more than aware. For starters, they'll be without a number of key players at the start of season due to World Cup qualifiers for Brazil and Australia, and Heath is once again sidelined by injury and may not return until as late as Week 4.
Foord's arrival in Portland has been delayed as she recovers from surgery to repair a ruptured Lisfanc ligament and unstable foot joint sustained during the W-League playoffs. Although the Thorns would likely have been without Foord until the conclusion of the Asian Cup, Australia's World Cup qualifiers, the team may now be without her until mid-season, although Parsons said that's likely "worst case scenario."
Whenever Foord does arrive in Portland, Parsons sees her not only filling in the scoring shoes of Nadim, but as being the attacker he feels the Thorns offense was lacking. Parsons has set the goal of being better offensively this year, and Foord is a player he says will help be just that.
"Wherever [Foord] plays—we see her in the front line for us—she can be one of the most dynamic forwards in this league and in a team that has been crying out for a dynamic forward up there to support Tobin and Sinc and Linds and the great work that has been going on so far with Hayley Raso."
During preseason Purce has also proven to be a player who could help finish out the Portland offense, especially in the beginning weeks while other players are missing.
"Midge Purce has been someone who is doing very, very well, and we're really happy," he said. "I would say that Midge Purce has been a massive addition, an important addition that I think impacts our style and lineup right off the bat."
On the other side of the ball, Parsons said that the goal is to maintain being the best defense in the league. That shouldn't be a problem, with Franch and defenders Katherine Reynolds, Emily Sonnett, Emily Menges, and Meghan Klingenberg all healthy and returning to the Thorns this year.
If fans were worried by the Thorns seemingly disappointing 0-2-1 record in preseason competition, Parsons is not. Instead, he's pleased with the performances put in and lessons learned from preseason camp and competition.
"Preseason has always been about growing and preparing for the game ahead at North Carolina and Chicago away and Orlando at home, and preparing and getting organized for that.
"We've done some good work so far, and we're going to continue that."
What the opponents think
The Equalizer has asked members of NWSL coaching staffs to comment on opposition clubs around the league. They were all promised anonymity.
"I think the Thorns will be not as good as everybody thinks. Henry, Nadim, [Long], you don't replace those players with [the players they have]. It depends when they get Tobin back. For the quality of players that Portland has, to play in a 5-3-2 and be as direct as they are, I find disappointing. They roll up their sleeves and they make it a fight and they come in and they battle with you. Credit to Mark for getting that level of player to be willing to play that kind of soccer, but they're capable of a lot more."
Related Topics2018 NWSL Season PreviewsAndressinhaCaitlin FoordFeaturedMark ParsonsTobin Heath
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When you disembark from the abra (water taxi) that takes you across brackish Dubai Creek and begin strolling the streets of the older Deira neighborhood, you'll soon enter the market district. A favorite stop here is the spice souk, which you'll smell at a distance of several blocks before you even enter it. It's a pleasant aroma, associated with beautiful and colorful displays. The spice souk adjoins the popular gold souk and, like it, is shaded by a high roof.
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Relaxing in a hot spring either tucked away in nature or in a more luxurious location is one of life's greatest pleasures. We've gathered our favorite hot spring locations in Washington state, ranging from family-friendly spots with lots of pampering to pristine wilderness with hikes included. Here are our favorite places throughout the state to take a relaxing dip!
The Olympic Hot Springs are an easy hike of about 5 mile round-trip alongside the Elwha River Valley. The dozen or so small pools are interspersed among trees with temps in the range of 100 to 112 degrees. These springs offer a more outdoorsy experience than the nearby Sol Duc Hot Springs Resort. Clothing is optional at these springs so just a heads up for family hot spring visitors. The trailhead is about 20 miles south of Port Angeles inside the Olympic National Park. The park entrance fee is $25 per vehicle, good for seven days. Camping is available nearby at the Boulder Creek Campground. Find more information at wta.org.
For the hot spring lovers who want a bit more of a luxurious experience, then Sol Duc Hot Springs is the idea location. Sol Duc is in the northwest corner of Olympic National Park, about 3 1/2 hours from Seattle. The three mineral soaking pools range in temperature from 99 to 104 degrees alongside a 50-degree freshwater pool. Other amenities include massage, an on-site restaurant and lodging in the on-site lodge, cabins or campground. There are also plenty of other activities to keep one busy from hikes in and around the Sol Duc Valley to Sol Duc Falls and Lake Crescent. For more information, visit nationalparkreservations.com.
Golymyer Hot Springs require a bit more effort to access but 75 mile drive (15 miles of which are on an non-maintained, unpaved Forest Service Road) from Seattle plus 5 mile hike are worth it when you spy the springs nestled in old-growth forest and nearby waterfalls. Temperatures range from 104 to 111 degrees at these springs nestled within the Alpine Lakes Wilderness. There is also a primitive campground nearby for overnight stays. Goldmyer Hot Springs is run by the nonprofit Northwest Wilderness program and reservations are required along with a Northwest Forest Pass and a high-clearance vehicle. Like Olympic Hot Springs, clothing is optional at these hot springs and reservations are limited to 20 people per day. For more information and to make reservations, visit goldmyer.org or call 206.789.5631.
Help keep public hot springs free and open to the public by obeying a few rules. Pack in and pack out your stuff. Leave alcohol at home along with glass bottles. And keep it clean both in behavior and by picking up after yourself.


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