Dual-specificity Tyrosine-regulated Kinase 1A Research Articles

DYRK1A controls the transition from proliferation to quiescence during lymphoid development by destabilizing Cyclin D3.

Pre-B and pre-T lymphocytes must orchestrate a transition from a highly proliferative state to a quiescent one during development. Cyclin D3 is essential for these cells' proliferation, but little is known about its posttranslational regulation at this stage. Here, we show that the dual specificity tyrosine-regulated kinase 1A (DYRK1A) restrains Cyclin D3 protein levels by phosphorylating T283 to induce its degradation. Loss of DYRK1A activity, via genetic inactivation or pharmacologic inhibition in mice, caused accumulation of Cyclin D3 protein, incomplete repression of E2F-mediated gene transcription, and failure to properly couple cell cycle exit with differentiation. Expression of a nonphosphorylatable Cyclin D3 T283A mutant recapitulated these defects, whereas inhibition of Cyclin D:CDK4/6 mitigated the effects of DYRK1A inhibition or loss. These data uncover a previously unknown role for DYRK1A in lymphopoiesis, and demonstrate how Cyclin D3 protein stability is negatively regulated during exit from the proliferative phases of B and T cell development.

The Chromosome 21 Kinase DYRK1A Controls Cell Cycle Exit and Survival During Lymphoid Development and Is a Novel Therapeutic Target In Acute Lymphoblastic Leukemia

The dual specificity tyrosine-regulated kinase 1A (DYRK1A) phosphorylates a growing number of proteins to influence various aspects of cell biology, including survival, apoptosis, proliferation, and neuronal development and function. Encoded on human chromosome 21, the DYRK1A locus resides in the Down syndrome critical region (DSCR) and has been implicated in the pathogenesis of neurocognitive deficits that result from trisomy 21. We recently demonstrated a prominent tumor-promoting role for DYRK1A in acute megakaryoblastic leukemia in children with DS (DS-AMKL). This discovery prompted us to ask whether DYRK1A is important for normal hematopoiesis, and if additional types of leukemia result from enhanced DYRK1A activity.Apart from our study in DS-AMKL, the involvement of DYRK1A in normal and malignant hematopoiesis has not been described. Dyrk1a germline knockout mice die mid-gestation; therefore, we generated conditional knockout (CKO) mice to allow inducible deletion of the kinase in hematopoietic cells with Mx1-Cre. In the bone marrow, CKO mice exhibit significant reductions of the hematopoietic stem cell-enriched LSK and common lymphoid progenitor populations (5-fold and >20-fold reductions vs. controls, respectively). In B cell lineage, CKO mice have 2-fold reductions of small pre-B and immature B cells, and a 5-fold reduction of mature recirculating B cells. In the thymus, CKO mice show dramatic loss of CD4+/CD8+double positive cells (DP, >30-fold fewer at 4 weeks). Furthermore, DYRK1A-deficient cells fail to reconstitute lymphoid cells in competitive bone marrow transplantation experiments and give rise to virtually no pre-B cell colonies in ex vivo assays. In contrast to these lymphoid defects, myeloid development is largely normal, with slightly increased numbers of erythroid precursors, granulocytes, and monocytes.To understand the mechanisms by which DYRK1A controls lymphopoiesis, we performed microwestern blot arrays. This approach allowed us to simultaneously screen >200 antibodies to identify differentially expressed proteins between CKO and control lymphoid cells. We found increased levels of cell cycle regulatory proteins, such as CDK1, suggesting that DYRK1A modulates cell cycle status during lymphoid development. Indeed, cell cycle analysis revealed that there were 20-30% fewer quiescent (G0) cells among CKO small pre-B cells and DP thymocytes, two developmental stages characterized by exit from the cell cycle following a proliferative burst. Among its targets, DYRK1A is known to contribute to DREAM (DP, Rb-like, E2F, and MuvB) complex assembly via phosphorylation of LIN52. The DREAM complex, not previously described in hematopoiesis, promotes quiescence by repressing the transcription of numerous cell cycle-associated genes during G0. RNA sequencing analysis in flow-purified G0 cells confirmed that DREAM complex targets are markedly de-repressed in the affected CKO populations, highlighting the importance of DYRK1A/DREAM complex interaction during lymphoid cell cycle exit.Both B- and T-lineage ALL express high levels of DYRK1A relative to other tumor types. Having observed that DYRK1A is essential for lymphoid cells, we next asked whether targeting DYRK1A with small molecule inhibitors would be an effective therapy for ALL. We found that EHT 1610, a highly selective DYRK1 inhibitor (Leblond B., Casagrande A.-S., Désiré L., Foucourt A., Besson T., DYRK1 inhibitors and uses thereof, WO 2013026806, 2013.02.28), dose-dependently induced apoptosis in B- and T-ALL cell lines and primary human pediatric ALL samples. Thus far, all cell lines and all primary samples tested were exquisitely sensitive to EHT 1610. Moreover, EHT 1610 induced apoptosis of primary ALL cells that were resistant to cytarabine, suggesting that DYRK1A inhibitors may be used in combination with standard ALL therapies for refractory or relapsed cases. Together, our data establish novel essential roles for DYRK1A in both normal and malignant lymphoid development and provide rationale for the design of DYRK1A-targeted ALL therapies. Disclosures:Leblond:Diaxonhit Therapeutics: Employment. Casagrande:Diaxonhit Therapeutics: Employment. Désiré:Diaxonhit Therapeutics: Employment. Crispino:Sanofi: Research Funding.

Dissection of the C-Terminal Region of E1A Redefines the Roles of CtBP and Other Cellular Targets in Oncogenic Transformation

Human adenovirus E1A makes extensive connections with the cellular protein interaction network. By doing so, E1A can manipulate many cellular programs, including cell cycle progression. Through these reprogramming events, E1A functions as a growth-promoting oncogene and has been used extensively to investigate mechanisms contributing to oncogenesis. Nevertheless, it remains unclear how the C-terminal region of E1A contributes to oncogenic transformation. Although this region is required for transformation in cooperation with E1B, it paradoxically suppresses transformation in cooperation with activated Ras. Previous analysis has suggested that the interaction of E1A with CtBP plays a pivotal role in both activities. However, some C-terminal mutants of E1A retain CtBP binding and yet exhibit defects in transformation, suggesting that other targets of this region are also necessary. To explore the roles of these additional factors, we performed an extensive mutational analysis of the C terminus of E1A. We identified key residues that are specifically required for binding all known targets of the C terminus of E1A. We further tested each mutant for the ability to both localize to the nucleus and transform primary rat cells in cooperation with E1B-55K or Ras. Interaction of E1A with importin α3/Qip1, dual-specificity tyrosine-regulated kinase 1A (DYRK1A), HAN11, and CtBP influenced transformation with E1B-55K. Interestingly, the interaction of E1A with DYRK1A and HAN11 appeared to play a role in suppression of transformation by activated Ras whereas interaction with CtBP was not necessary. This unexpected result suggests a need for revision of current models and provides new insight into transformation by the C terminus of E1A.

Development of a Mouse Model of DS Megakaryoblastic Leukemia and Identification of the Hsa21 Gene DYRK1A as a Prominent Contributor to DS-AMKL

AbstractAbstract 469Children with Down syndrome (DS) have a 500-fold increased risk of Acute Megakaryoblastic Leukemia (DS-AMKL). In addition to trisomy 21, DS-AMKL blasts harbor somatic mutations in the gene encoding GATA-1, an essential transcriptional regulator of erythroid and megakaryocytic differentiation. In every case, the GATA1 mutations lead to a block in expression of the full-length protein, but allow for the translation of a shortened isoform named GATA-1s, which lacks the N-terminal transcriptional activation domain. Recent studies have shown that mutations in of JAK2, JAK3 and MPL are another feature of AMKL. Whereas the impact on hematopoiesis of individual GATA-1s, JAK2 V617F and MPL W515L mutants is relatively well known, the specific effects of trisomy 21, the initiating event in DS leukemogenesis, remains unclear. Here, we used two different experimental models to identify specific genes on human chromosome 21 (Hsa21) that participate in leukemogenesis: human DS-AMKL cell lines and the Ts1Rhr mouse strain, which is trisomic for only 33 orthologs of Hsa21 genes. Comprehensive analysis of hematopoiesis in progressively more complex mouse models of DS disorders, including Ts1Rhr mice, Ts1Rhr/KI-Gata-1s compound mutant mice, and recipients of Ts1Rhr/KI-Gata-1s hematopoietic progenitors expressing MPL W515L, revealed that the presence of trisomy 21 impacts the phenotype of each genetic background. Ts1Rhr mice develop a progressive myeloproliferative disease with megakaryocytic features while Ts1Rhr/KI-Gata1s double transgenic mice develop thrombocytosis at an earlier age compared to Gata1s mutant or Ts1Rhr mice alone. However neither the Ts1Rhr nor the compound mutant mice develop AMKL. In stark contrast, expression of MPL W515L in Ts1Rhr/KI-Gata1s hematopoietic progenitors led to a rapid lethal megakaryoblastic disorder characterized by profound marrow and splenic fibrosis and the presence of immature megakaryoblasts in lethally irradiated recipients. Of note, transplantation of MPL W515L expressing singly mutant Ts1Rhr or KI-Gata-1s progenitors failed to cause a similar disease. These findings show that three oncogenic events are necessary and sufficient to induce a megakaryoblastic leukemia in recipient mice. In parallel, to identify specific genes on Hsa21 that contribute to AMKL, we performed shRNA screening in DS-AMKL cell lines by knocking down the human orthologs of the 33 genes trisomic in the Ts1Rhr mice. We then evaluated the consequence of knockdown on megakaryocyte proliferation, survival and/or differentiation. This study revealed that altered expression of ERG, DYRK1A, CHAF1B and HLCS affected megakaryocyte growth. Analysis of their expression in human samples suggest that these four genes likely play a role in etiology of DS-AMKL. Moreover expression evaluation of those genes in our in vivo models of 1, 2 or 3 genetic events revealed that ERG, a known megakaryoblastic oncogene, and DYRK1A are the most prominent genes implicated in these megakaryocytic disorders. DYRK1A (dual specificity tyrosine-regulated kinase 1A) has been functionally implicated in many developmental disorders associated with DS and shown to be constitutively phosphorylated/activated in human AMKL cell lines. We found that overexpression of DYRK1A cooperates with Gata1s to lead to a increase in megakaryocyte growth in liquid culture and in colony assays in vitro. In summary, we show that partially trisomic Ts1Rhr mice display progressive defects in the megakaryocytic compartment, that mutagenesis of Gata1 in the Ts1Rhr background exacerbates the Ts1Rhr phenotype, and that addition of an activating mutation of MPL leads to a fulminant megakaryocytic leukemia with myelofibrosis. Moreover, among the 33 genes present in 3 copies in the Ts1Rhr background, we identify DYRK1A as a likely megakaryoblastic oncogene in DS-AMKL. Disclosures:No relevant conflicts of interest to declare.

Age-associated motor and visuo-spatial learning phenotype in Dyrk1A heterozygous mutant mice

Dual-specificity tyrosine-regulated kinase 1A ( DYRK1A) is a candidate gene for the Down syndrome neurological defects and may be involved in the progression of Alzheimer's disease. Heterozygous mice for Dyrk1A (Dyrk1A+/−) exhibit decreased brain size, motor abnormalities and cognitive deficits in the adult. However, there is no information about the mutant phenotype in old ages. Here we analyze the impact of Dyrk1A dosage reduction on motor performance and hippocampal-dependent learning and memory in aged Dyrk1A+/− mice. Whereas motor tests showed marked alterations in traction ability, prehensile reflex and balance, heterozygous mice only present a slight impairment of visuo-spatial memory even though they show a robust decrease of CA1–CA3 and dentate gyrus cells.

The DYRK1A gene, encoded in chromosome 21 Down syndrome critical region, bridges between β-amyloid production and tau phosphorylation in Alzheimer disease

We scanned throughout chromosome 21 to assess genetic associations with late-onset Alzheimer disease (AD) using 374 Japanese patients and 375 population-based controls, because trisomy 21 is known to be associated with early deposition of beta-amyloid (Abeta) in the brain. Among 417 markers spanning 33 Mb, 22 markers showed associations with either the allele or the genotype frequency (P < 0.05). Logistic regression analysis with age, sex and apolipoprotein E (APOE)-epsilon4 dose supported genetic risk of 17 markers, of which eight markers were linked to the SAMSN1, PRSS7, NCAM2, RUNX1, DYRK1A and KCNJ6 genes. In logistic regression, the DYRK1A (dual-specificity tyrosine-regulated kinase 1A) gene, located in the Down syndrome critical region, showed the highest significance [OR = 2.99 (95% CI: 1.72-5.19), P = 0.001], whereas the RUNX1 gene showed a high odds ratio [OR = 23.3 (95% CI: 2.76-196.5), P = 0.038]. DYRK1A mRNA level in the hippocampus was significantly elevated in patients with AD when compared with pathological controls (P < 0.01). DYRK1A mRNA level was upregulated along with an increase in the Abeta-level in the brain of transgenic mice, overproducing Abeta at 9 months of age. In neuroblastoma cells, Abeta induced an increase in the DYRK1A transcript, which also led to tau phosphorylation at Thr212 under the overexpression of tau. Therefore, the upregulation of DYRK1A transcription results from Abeta loading, further leading to tau phosphorylation. Our result indicates that DYRK1A could be a key molecule bridging between beta-amyloid production and tau phosphorylation in AD.

Dyrk1A Phosphorylates α-Synuclein and Enhances Intracellular Inclusion Formation

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.

Constitutive Dyrk1A is abnormally expressed in Alzheimer disease, Down syndrome, Pick disease, and related transgenic models

DYRK1A, dual-specificity tyrosine-regulated kinase 1A, maps to human chromosome 21 within the Down syndrome (DS) critical region. Dyrk1 phosphorylates the human microtubule-associated protein tau at Thr212 in vitro, a residue that is phosphorylated in fetal tau and hyper-phosphorylated in Alzheimer disease (AD) and tauopathies, including Pick disease (PiD). Furthermore, phosphorylation of Thr212 primes tau for phosphorylation by glycogen synthase kinase 3 (GSK-3). The present study examines Dyrk1A in the cerebral cortex of sporadic AD, adult DS with associated AD, and PiD. Increased Dyrk1A immunoreactivity has been found in the cytoplasm and nuclei of scattered neurons of the neocortex, entorhinal cortex, and hippocampus in AD, DS, and PiD. Dyrk1A is found in sarkosyl-insoluble fractions which are enriched in phosphorylated tau in AD brains, thus suggesting a possible association of Dyrk1A with neurofibrillary tangle pathology. Yet, no clear relationship has been observed between tau phosphorylation at Thr212, and GSK-3 and Dyrk1A expression in diseased brains. Transgenic mice bearing a triple tau mutation (G272V, P301L, and R406W) and expressing hyper-phosphoyrylated tau in neurons of the entorhinal cortex, hippocampus, and cerebral neocortex show increased expression of Dyrk1A in individual neurons in the same regions. However, transgenic mice over-expressing Dyrk1A do not show increased phosphorylation of tau at Thr212, thus suggesting that Dyrk1A over-expression does not trigger per se hyper-phosphorylation of tau at Thr212 in vivo. The present observations indicate modifications in the expression of constitutive Dyrk1A in the cytoplasm and nuclei of neurons in various neurodegenerative diseases associated with tau phosphorylation.