DREAM On: Cell Cycle Control in Development and Disease.

Perfectly orchestrated periodic gene expression during cell cycle progression is essential for maintaining genome integrity and ensuring that cell proliferation can be stopped by environmental signals. Genetic and proteomic studies during the past two decades revealed remarkable evolutionary conservation of the key mechanisms that control cell cycle-regulated gene expression, including multisubunit DNA-binding DREAM complexes. DREAM complexes containing a retinoblastoma family member, an E2F transcription factor and its dimerization partner, and five proteins related to products of Caenorhabditis elegans multivulva (Muv) class B genes lin-9, lin-37, lin-52, lin-53, and lin-54 (comprising the MuvB core) have been described in diverse organisms, from worms to humans. This review summarizes the current knowledge of the structure, function, and regulation of DREAM complexes in different organisms, as well as the role of DREAM in human disease.

PAF remodels the DREAM complex to bypass cell quiescence and promote lung tumorigenesis.

The DREAM complex orchestrates cell quiescence and the cell cycle. However, how the DREAM complex is deregulated in cancer remains elusive. Here, we report that PAF (PCLAF/KIAA0101) drives cell quiescence exit to promote lung tumorigenesis by remodeling the DREAM complex. PAF is highly expressed in lung adenocarcinoma (LUAD) and is associated with poor prognosis. Importantly, Paf knockout markedly suppressed LUAD development in mouse models. PAF depletion induced LUAD cell quiescence and growth arrest. PAF is required for the global expression of cell-cycle genes controlled by the repressive DREAM complex. Mechanistically, PAF inhibits DREAM complex formation by binding to RBBP4, a core DREAM subunit, leading to transactivation of DREAM target genes. Furthermore, pharmacological mimicking of PAF-depleted transcriptomes inhibited LUAD tumor growth. Our results unveil how the PAF-remodeled DREAM complex bypasses cell quiescence to promote lung tumorigenesis and suggest that the PAF-DREAM axis may be a therapeutic vulnerability in lung cancer.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function variations in the dual specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein were expressed throughout the developing head and both were enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with variants of DYRK1A.

Structural mechanisms of DREAM complex assembly and regulation.

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52-p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence.

PP2A-mediated regulation of Ras signaling in G2 is essential for stable quiescence and normal G1 length.

Quiescence (G0) allows cycling cells to reversibly cease proliferation. A decision to enter quiescence is suspected of occurring early in G1, before the restriction point (R). Surprisingly, we have identified G2 as an interval during which inhibition of the protein phosphatase PP2A results in failure to exhibit stable quiescence. This effect is accompanied by shortening of the ensuing G1. The PP2A subcomplex required for stable G0 contains the B56γ B subunit. After PP2A inhibition in G2, aberrant overexpression of cyclin E occurs during mitosis and is responsible for overriding quiescence. Strikingly, suppression of Ras signaling re-establishes normal cyclin E levels during M and restores G0. These data point to PP2A-B56γ-driven Ras signaling modulation in G2 as essential for suppressing aberrant cyclin E expression during mitosis and thereby achieving normal G0 control. Thus, G2 is an interval during which the length and growth factor dependence of the next G1 interval are established.

Structural mechanism of Myb-MuvB assembly.

The MuvB transcriptional regulatory complex, which controls cell-cycle-dependent gene expression, cooperates with B-Myb to activate genes required for the G2 and M phases of the cell cycle. We have identified the domain in B-Myb that is essential for the assembly of the Myb-MuvB (MMB) complex. We determined a crystal structure that reveals how this B-Myb domain binds MuvB through the adaptor protein LIN52 and the scaffold protein LIN9. The structure and biochemical analysis provide an understanding of how oncogenic B-Myb is recruited to regulate genes required for cell-cycle progression, and the MMB interface presents a potential therapeutic target to inhibit cancer cell proliferation.

Proteomic Landscape of Tissue-Specific Cyclin E Functions in Vivo.

E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their 'core' cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer.

DYRK1A protein kinase promotes quiescence and senescence through DREAM complex assembly.

In the absence of growth signals, cells exit the cell cycle and enter into G0 or quiescence. Alternatively, cells enter senescence in response to inappropriate growth signals such as oncogene expression. The molecular mechanisms required for cell cycle exit into quiescence or senescence are poorly understood. The DREAM (DP, RB [retinoblastoma], E2F, and MuvB) complex represses cell cycle-dependent genes during quiescence. DREAM contains p130, E2F4, DP1, and a stable core complex of five MuvB-like proteins: LIN9, LIN37, LIN52, LIN54, and RBBP4. In mammalian cells, the MuvB core dissociates from p130 upon entry into the cell cycle and binds to BMYB during S phase to activate the transcription of genes expressed late in the cell cycle. We used mass spectroscopic analysis to identify phosphorylation sites that regulate the switch of the MuvB core from BMYB to DREAM. Here we report that DYRK1A can specifically phosphorylate LIN52 on serine residue 28, and that this phosphorylation is required for DREAM assembly. Inhibiting DYRK1A activity or point mutation of LIN52 disrupts DREAM assembly and reduces the ability of cells to enter quiescence or undergo Ras-induced senescence. These data reveal an important role for DYRK1A in the regulation of DREAM activity and entry into quiescence.

A kinase shRNA screen links LATS2 and the pRB tumor suppressor.

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.

Interpreting cancer genomes using systematic host network perturbations by tumour virus proteins.

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.

Coordinated repression of cell cycle genes by KDM5A and E2F4 during differentiation.

Epigenetic regulation underlies the robust changes in gene expression that occur during development. How precisely epigenetic enzymes contribute to development and differentiation processes is largely unclear. Here we show that one of the enzymes that removes the activating epigenetic mark of trimethylated lysine 4 on histone H3, lysine (K)-specific demethylase 5A (KDM5A), reinforces the effects of the retinoblastoma (RB) family of transcriptional repressors on differentiation. Global location analysis showed that KDM5A cooccupies a substantial portion of target genes with the E2F4 transcription factor. During ES cell differentiation, knockout of KDM5A resulted in derepression of multiple genomic loci that are targets of KDM5A, denoting a direct regulatory function. In terminally differentiated cells, common KDM5A and E2F4 gene targets were bound by the pRB-related protein p130, a DREAM complex component. KDM5A was recruited to the transcription start site regions independently of E2F4; however, it cooperated with E2F4 to promote a state of deepened repression at cell cycle genes during differentiation. These findings reveal a critical role of H3K4 demethylation by KDM5A in the transcriptional silencing of genes that are suppressed by RB family members in differentiated cells.

Identification of FAM111A as an SV40 host range restriction and adenovirus helper factor.

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

The CHR promoter element controls cell cycle-dependent gene transcription and binds the DREAM and MMB complexes.

Cell cycle-dependent gene expression is often controlled on the transcriptional level. Genes like cyclin B, CDC2 and CDC25C are regulated by cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) promoter elements mainly through repression in G(0)/G(1). It had been suggested that E2F4 binding to CDE sites is central to transcriptional regulation. However, some promoters are only controlled by a CHR. We identify the DREAM complex binding to the CHR of mouse and human cyclin B2 promoters in G(0). Association of DREAM and cell cycle-dependent regulation is abrogated when the CHR is mutated. Although E2f4 is part of the complex, a CDE is not essential but can enhance binding of DREAM. We show that the CHR element is not only necessary for repression of gene transcription in G(0)/G(1), but also for activation in S, G(2) and M phases. In proliferating cells, the B-myb-containing MMB complex binds the CHR of both promoters independently of the CDE. Bioinformatic analyses identify many genes which contain conserved CHR elements in promoters binding the DREAM complex. With Ube2c as an example from that screen, we show that inverse CHR sites are functional promoter elements that can bind DREAM and MMB. Our findings indicate that the CHR is central to DREAM/MMB-dependent transcriptional control during the cell cycle.

Dyrk1a is required for craniofacial development in Xenopus laevis.

Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis . Dyrk1a mRNA and protein was expressed throughout the developing head and was enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with DYRK1A mutations.

Proteasome inhibition paradoxically degrades gain-of-function mutant p53 R273H in NSCLC and could have therapeutic implications.

Lung cancer is the leading cause of cancer mortality. Despite therapeutic advances in recent years, new treatment strategies are needed to improve outcomes of lung cancer patients. Mutant p53 is prevalent in lung cancers and drives several hallmarks of cancer through a gain-of-function oncogenic program, and often predicts a poorer prognosis. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. In this study, we report that proteasome inhibition results in degradation of mutant p53 in non-small cell lung cancer (NSCLC) cell lines bearing the R273H mutant protein and show evidence that this was mediated by hsp70. NSCLC cell lines with the mutant R273H allele demonstrated increased susceptibility and apoptosis to proteasome inhibitors. These data suggest that proteasome inhibitors could have therapeutic implications in some subsets of TP53 mutated NSCLC.

Insights from the protein interaction Universe of the multifunctional "Goldilocks" kinase DYRK1A.

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is encoded by a dosage-dependent gene located in the Down syndrome critical region of human chromosome 21. The known substrates of DYRK1A include proteins involved in transcription, cell cycle control, DNA repair and other processes. However, the function and regulation of this kinase is not fully understood, and the current knowledge does not fully explain the dosage-dependent function of this kinase. Several recent proteomic studies identified DYRK1A interacting proteins in several human cell lines. Interestingly, several of known protein substrates of DYRK1A were undetectable in these studies, likely due to a transient nature of the kinase-substrate interaction. It is possible that the stronger-binding DYRK1A interacting proteins, many of which are poorly characterized, are involved in regulatory functions by recruiting DYRK1A to the specific subcellular compartments or distinct signaling pathways. Better understanding of these DYRK1A-interacting proteins could help to decode the cellular processes regulated by this important protein kinase during embryonic development and in the adult organism. Here, we review the current knowledge of the biochemical and functional characterization of the DYRK1A protein-protein interaction network and discuss its involvement in human disease.

Exploitation of Sulfated Glycosaminoglycan Status for Precision Medicine of Triplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking targetable biomarkers. TNBC is known to be most aggressive and when metastatic is often drug-resistant and uncurable. Biomarkers predicting response to therapy improve treatment decisions and allow personalized approaches for patients with TNBC. This study explores sulfated glycosaminoglycan (sGAG) levels as a predictor of TNBC response to platinum therapy. sGAG levels were quantified in three distinct TNBC tumor models, including cell line-derived, patient-derived xenograft (PDX) tumors, and isogenic models deficient in sGAG biosynthesis. The in vivo antitumor efficacy of Triplatin, a sGAG-directed platinum agent, was compared in these models with the clinical platinum agent, carboplatin. We determined that >40% of TNBC PDX tissue microarray samples have high levels of sGAGs. The in vivo accumulation of Triplatin in tumors as well as antitumor efficacy of Triplatin positively correlated with sGAG levels on tumor cells, whereas carboplatin followed the opposite trend. In carboplatin-resistant tumor models expressing high levels of sGAGs, Triplatin decreased primary tumor growth, reduced lung metastases, and inhibited metastatic growth in lungs, liver, and ovaries. sGAG levels served as a predictor of Triplatin sensitivity in TNBC. Triplatin may be particularly beneficial in treating patients with chemotherapy-resistant tumors who have evidence of residual disease after standard neoadjuvant chemotherapy. More effective neoadjuvant and adjuvant treatment will likely improve clinical outcome of TNBC.

Simultaneous expression of MMB-FOXM1 complex components enables efficient bypass of senescence.

Cellular senescence is a stable cell cycle arrest that normal cells undergo after a finite number of divisions, in response to a variety of intrinsic and extrinsic stimuli. Although senescence is largely established and maintained by the p53/p21WAF1/CIP1 and pRB/p16INK4A tumour suppressor pathways, the downstream targets responsible for the stability of the growth arrest are not known. We have employed a stable senescence bypass assay in conditionally immortalised human breast fibroblasts (CL3EcoR) to investigate the role of the DREAM complex and its associated components in senescence. DREAM is a multi-subunit complex comprised of the MuvB core, containing LIN9, LIN37, LIN52, LIN54, and RBBP4, that when bound to p130, an RB1 like protein, and E2F4 inhibits cell cycle-dependent gene expression thereby arresting cell division. Phosphorylation of LIN52 at Serine 28 is required for DREAM assembly. Re-entry into the cell cycle upon phosphorylation of p130 leads to disruption of the DREAM complex and the MuvB core, associating initially to B-MYB and later to FOXM1 to form MMB and MMB-FOXM1 complexes respectively. Here we report that simultaneous expression of MMB-FOXM1 complex components efficiently bypasses senescence with LIN52, B-MYB, and FOXM1 as the crucial components. Moreover, bypass of senescence requires non-phosphorylated LIN52 that disrupts the DREAM complex, thereby indicating a central role for assembly of the DREAM complex in senescence.

Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Progesterone Receptors Promote Quiescence and Ovarian Cancer Cell Phenotypes via DREAM in p53-Mutant Fallopian Tube Models.

CONTEXT: The ability of ovarian steroids to modify ovarian cancer (OC) risk remains controversial. Progesterone is considered to be protective; recent studies indicate no effect or enhanced OC risk. Knowledge of progesterone receptor (PR) signaling during altered physiology that typifies OC development is limited. OBJECTIVE: This study defines PR-driven oncogenic signaling mechanisms in p53-mutant human fallopian tube epithelia (hFTE), a precursor of the most aggressive OC subtype. METHODS: PR expression in clinical samples of serous tubal intraepithelial carcinoma (STIC) lesions and high-grade serous OC (HGSC) tumors was analyzed. Novel PR-A and PR-B isoform-expressing hFTE models were characterized for gene expression and cell cycle progression, emboli formation, and invasion. PR regulation of the DREAM quiescence complex and DYRK1 kinases was established. RESULTS: STICs and HGSC express abundant activated phospho-PR. Progestin promoted reversible hFTE cell cycle arrest, spheroid formation, and invasion. RNAseq/biochemical studies revealed potent ligand-independent/-dependent PR actions, progestin-induced regulation of the DREAM quiescence complex, and cell cycle target genes through enhanced complex formation and chromatin recruitment. Disruption of DREAM/DYRK1s by pharmacological inhibition, HPV E6/E7 expression, or DYRK1A/B depletion blocked progestin-induced cell arrest and attenuated PR-driven gene expression and associated OC phenotypes. CONCLUSION: Activated PRs support quiescence and pro-survival/pro-dissemination cell behaviors that may contribute to early HGSC progression. Our data support an alternative perspective on the tenet that progesterone always confers protection against OC. STICs can reside undetected for decades prior to invasive disease; our studies reveal clinical opportunities to prevent the ultimate development of HGSC by targeting PRs, DREAM, and/or DYRKs.