Dual-specificity Tyrosine Phosphorylation-regulated Kinase Family Research Articles

Dual-specificity kinase DYRK3 phosphorylates p62 at the Thr-269 residue and promotes melanoma progression

Melanoma is a type of skin cancer that originates in melanin-producing melanocytes. It is considered a multifactorial disease caused by both genetic and environmental factors, such as UV radiation. Dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) phosphorylates many substrates involved in signaling pathways, cell survival, cell cycle control, differentiation, and neuronal development. However, little is known about the cellular function of DYRK3, one of the five members of the DYRK family. Interestingly, it was observed that the expression of DYRK3, as well as p62 (a multifunctional signaling protein), is highly enhanced in most melanoma cell lines. This study aimed to investigate whether DYRK3 interacts with p62, and how this affects melanoma progression, particularly in melanoma cell lines. We found that DYRK3 directly phosphorylates p62 at the Ser-207 and Thr-269 residue. Phosphorylation at Thr-269 of p62 by DYRK3 increased the interaction of p62 with TRAF6, an already known activator of mTORC1 in the mTOR-involved signaling pathways. Moreover, the phosphorylation of p62 at Thr-269 promoted the activation of mTORC1. We also found that DYRK3-mediated phosphorylation of p62 at Thr-269 enhanced the growth of melanoma cell lines and melanoma progression. Conversely, DYRK3 knockdown or blockade of p62-T269 phosphorylation inhibited melanoma growth, colony formation, and cell migration. In conclusion, we demonstrated that DYRK3 phosphorylates p62, positively modulating the p62-TRAF6-mTORC1 pathway in melanoma cells. This finding suggests that DYRK3 suppression may be a novel therapy for preventing melanoma progression by regulating the mTORC1 pathway.

Straightforward Access to a New Class of Dual DYRK1A/CLK1 Inhibitors Possessing a Simple Dihydroquinoline Core.

The DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) family of protein kinases is involved in the pathogenesis of several neurodegenerative diseases. Among them, the DYRK1A protein kinase is thought to be implicated in Alzheimer's disease (AD) and Down syndrome, and as such, has emerged as an appealing therapeutic target. DYRKs are a subset of the CMGC (CDK, MAPKK, GSK3 and CLK) group of kinases. Within this group of kinases, the CDC2-like kinases (CLKs), such as CLK1, are closely related to DYRKs and have also sparked great interest as potential therapeutic targets for AD. Based on inhibitors previously described in the literature (namely TG003 and INDY), we report in this work a new class of dihydroquinolines exhibiting inhibitory activities in the nanomolar range on hDYRK1A and hCLK1. Moreover, there is overwhelming evidence that oxidative stress plays an important role in AD. Pleasingly, the most potent dual kinase inhibitor 1p exhibited antioxidant and radical scavenging properties. Finally, drug-likeness and molecular docking studies of this new class of DYRK1A/CLK1 inhibitors are also discussed in this article.

Roles of dual specificity tyrosine-phosphorylation-regulated kinase 2 in nervous system development and disease.

Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are a group of conserved eukaryotic kinases phosphorylating tyrosine, serine, and threonine residues. The human DYRK family comprises 5 members (DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4). The different DYRKs have been implicated in neurological diseases, cancer, and virus infection. Specifically, DYRK2 has been mainly implicated in cancer progression. However, its role in healthy and pathological nervous system function has been overlooked. In this context, we review current available data on DYRK2 in the nervous system, where the available studies indicate that it has key roles in neuronal development and function. DYRK2 regulates neuronal morphogenesis (e.g., axon growth and branching) by phosphorylating cytoskeletal elements (e.g., doublecortin). Comparative data reveals that it is involved in the development of olfactory and visual systems, the spinal cord and possibly the cortex. DYRK2 also participates in processes such as olfaction, vision and, learning. However, DYRK2 could be involved in other brain functions since available expression data shows that it is expressed across the whole brain. High DYRK2 protein levels have been detected in basal ganglia and cerebellum. In adult nervous system, DYRK2 mRNA expression is highest in the cortex, hippocampus, and retina. Regarding nervous system disease, DYRK2 has been implicated in neuroblastoma, glioma, epilepsy, neuroinflammation, Alzheimer’s disease, Parkinson’s disease, spinal cord injury and virus infection. DYRK2 upregulation usually has a negative impact in cancer-related conditions and a positive impact in non-malignant conditions. Its role in axon growth makes DYRK2 as a promising target for spinal cord or brain injury and regeneration.

Suppression of DYRK1A/B Drives Endoplasmic Reticulum Stress-mediated Autophagic Cell Death Through Metabolic Reprogramming in Colorectal Cancer Cells.

We previously identified KS40008 (4-(3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzene-1,2-diol), a novel inhibitor of dual-specificity tyrosine phosphorylation-regulated kinase family (DYRK) 1A/B, which exhibited high enzymatic activity and cell proliferation-inhibitory effects in colorectal cancer (CRC) cell lines. In the present study, we aimed to elucidate the antitumor mechanisms of KS40008. To assess the cytotoxicity of KS40008, we utilized a human cell line and organoid model and performed a CCK-8 assay and real-time cell analysis. Mitochondrial function was determined through mitochondrial staining, mito-stress test, and glycolysis test. In addition, we investigated the mechanisms of cancer cell death induced by KS40008 through immunoblotting, real-time quantitative polymerase chain reaction, reactive oxygen species staining, and immunofluorescence staining. KS40008 exhibited significant cytotoxicity in CRC and non-CRC cell lines, and organoid models compared to 5-fluorouracil, a conventional chemotherapeutic drug. Moreover, KS40008-induced inhibition of DYRK1A/B led to mitochondrial dysfunction and endoplasmic reticulum stress, promoting autophagic cancer cell death. KS40008 exerts antitumor activity through the inhibition of DYRK1A/B. Here, we demonstrated a mechanism by which KS40008 affects endoplasmic reticulum stress-mediated autophagy through the induction of mitochondrial stress, leading to cytotoxicity in CRC.

Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition

Background: KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 75% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified that DYRK1A is specifically required for the survival of KMT2A-R ALL cell. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as negatively regulator of cell proliferation.Results: We performed a kinome-wide CRISPR screen in human ALL cell lines and PDX models and identified DYRK1A as a novel target in KMT2A-R ALL. DYRK1A is a serine-threonine kinase with a proposed, but poorly defined role in cell cycle regulation. We performed a meta-analysis of multiple ChIP-Seq experiments and identified that oncogenic KMT2A fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses of KMT2A-R ALL cells treated with a menin inhibitor (MI-503) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A, indicating that DYRK1A is directly regulated by the KMT2A fusion complex. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced potent leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells.Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. [Display omitted] DisclosuresCrispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.

A plant-specific DYRK kinase DYRKP coordinates cell morphology in Marchantia polymorpha

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) are activated via the auto-phosphorylation of conserved tyrosine residues in their activation loop during protein translation, and they then phosphorylate serine/threonine residues on substrates. The DYRK family is widely conserved in eukaryotes and is composed of six subgroups. In plant lineages, DYRK homologs are classified into four subgroups, DYRK2s, yet another kinase1s, pre-mRNA processing factor 4 kinases, and DYRKPs. Only the DYRKP subgroup is plant-specific and has been identified in a wide array of plant lineages, including land plants and green algae. It has been suggested that in Arabidopsis thaliana DYRKPs are involved in the regulation of centripetal nuclear positioning induced by dark light conditions. However, the molecular functions, such as kinase activity and the developmental and physiological roles of DYRKPs are poorly understood. Here, we focused on a sole DYRKP ortholog in the model bryophyte, Marchantia polymorpha, MpDYRKP. MpDYRKP has a highly conserved kinase domain located in the C-terminal region and shares common sequence motifs in the N-terminal region with other DYRKP members. To identify the roles of MpDYRKP in M. polymorpha, we generated loss-of-function Mpdyrkp mutants via genome editing. Mpdyrkp mutants exhibited abnormal, shrunken morphologies with less flattening in their vegetative plant bodies, thalli, and male reproductive organs, antheridial receptacles. The surfaces of the thalli in the Mpdyrkp mutants appeared uneven and disordered. Moreover, their epidermal cells were drastically altered to a narrower shape when compared to the wild type. These results suggest that MpDYRKP acts as a morphological regulator, which contributes to orderly tissue morphogenesis via the regulation of cell shape.

DYRK1A Is Required to Alleviate Replication Stress in KMT2A-Rearranged Acute Lymphoblastic Leukemia

Background: KMT2A-rearranged (R) ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in children and adults with ALL. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified DYRK1A as required for KMT2A-R ALL cell survival, but not in other high risk ALL genetic subtypes. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncoprotein in a murine Down syndrome model of megakaryoblastic leukemia. DYRK1A negatively regulates cell proliferation and induces quiescence. Paradoxically, genetic deletion or pharmacological inhibition of DYRK1A upregulates the cell cycle regulator CCND3 and increased numbers of B cells in S-phase, yet also significantly reduces cell proliferation. The specific role of DYRK1A in ALL has not been reported. Results: We assessed the importance of DYRK1A deletion in a focused screen of 14 previously identified kinases. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines and a human KMT2A-Aff1-FLAG transduced ALL model demonstrated direct binding of both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-fusion to the DYRK1A promoter. To assess if KMT2A fusion directly regulates DYRK1A expression, we treated SEM cells with the menin-KMT2A disrupter MI-503 and identified that the KMT2A fusion protein is a positive regulator of DYRK1A. Pharmacologic inhibition of DYRK1A with EHT1610 demonstrated potent leukemic cell growth inhibition, demonstrating that DYRK1 could be a new therapeutic target in KMT2A-R ALL. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which resulted in accumulation of CCND3 as expected. In addition, we detected upregulation of the positive cell cycle regulator MYC and the replication stress response molecule CHK1. In a second experiment, we validated the upregulation of MYC and identified significant upregulation of the proapoptotic protein BIM. Strikingly, meta-analysis of gene expression data from Dyrk1a-deleted murine pre-B cells isolated from a conditional Dyrk1a knockout mouse model also demonstrated increased levels of MYC and CHK1, validating that the EHT1610 mediated upregulation of MYC or CHK1 is a specific effect induced by DYRK1A inhibition. Western blot analysis demonstrated that KMT2A-R ALL cell lines have constitutive activation of pH2AX. Based on these data, we hypothesize that DYRK1A-mediated upregulation of CCND3 and MYC forces the cells to proliferate, which significantly increases replication stress and causes apoptosis, as evident by upregulation of CHK1 and BIM. To test if targeting the interaction of BIM with BCL2 will have an increased apoptotic effect when combined with EHT1610, we treated two KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, we observed a synergistic effect with both drugs, suggesting that combining these inhibitors has superior anti-leukemic activity. Conclusions: DYRK1A and MYC are positively regulated by the KMT2A fusion protein in KMT2A-R ALL and negatively regulate each other. Pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells due to increased MYC and CHK1 proteins that induce replication stress. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for KMT2A-R ALL that is translatable to the clinic for patients with these high-risk leukemias. Disclosures Tasian: Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Research Funding.

DYRK1A Is Regulated By Oncogenic KMT2A and Required for Survival of KMT2A-Rearranged Acute Lymphoblastic Leukemia

Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.

Abstract 1902: Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma

Abstract The purpose of this study was to determine the effect of the natural product Harmine in human neuroblastoma (NB). NB is an early childhood malignancy arising from the developing peripheral nervous system. In up to 25% of cases MYCN gene amplification is correlated to high tumor stage and poor patient prognosis. High stage NB relapses frequently in spite of multimodal therapy and is then virtually untreatable. There is a clear need for specific, novel therapeutics. Harmine is a tricyclic β-carboline alkaloid from the harmal plant with cytostatic and cytotoxic effects on tumor cells. It is capable of blocking the activity of mitogen activate protein kinase (MAPK) and the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK) family proteins. These kinases inhibit apoptosis and encourage proliferation. Four human NB cell lines were used to study the effects of Harmine treatment: SK-N-BE(2)-C and KELLY (MYCN amplified) as well as SK-N-AS and SK-N-FI (MYCN single copy). Molecular interaction models of Harmine bound to DYRK family kinases were generated by computational docking using x-ray structures. The anti-cancer properties of Harmine were analyzed by real-time cell viability assays, in a dose-dependent manner, over a 72 hour period. The IC50 values were 169.9, 170.8, and 791.7 μM for SK-N-BE(2)-C, KELLY, and SK-N-FI, respectively, after 72 hours. Apoptosis assays for caspase activation, PARP cleavage, and annexin V induction, were performed using Western blot and flow cytometry. NB cell line exposure to 100 μM Harmine resulted in caspase-3/7 and caspase 9 activation, PARP cleavage, and annexin V-positive stained cells as early as 24 hours after treatment, indicating apoptosis induction. These results led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that Harmine induces NB cell death through a cellular mechanism that involves DYRK family kinases and triggers caspase-mediated apoptosis. Citation Format: Katie L. Uhl, Chad R. Schultz, Dirk Geerts, Andre S. Bachmann. Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1902.

Harmine, a dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) inhibitor induces caspase-mediated apoptosis in neuroblastoma

BackgroundNeuroblastoma (NB) is an early childhood malignancy that arises from the developing sympathetic nervous system. Harmine is a tricyclic β-carboline alkaloid isolated from the harmal plant that exhibits both cytostatic and cytotoxic effects. Harmine is capable of blocking the activities of dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) family proteins and mitogen-activated protein kinase. These kinases promote proliferation and inhibit apoptosis.MethodsFour human NB cell lines were used to study the effects of harmine treatment: SKNBE and KELLY (MYCN-amplified) as well as SKNAS and SKNFI (MYCN non-amplified). The anti-cancer properties of harmine were examined by RealTime-Glo MT cell viability assays, caspase activity assays, PARP cleavage using Western blot analysis, and flow cytometry-based Annexin V detection. A molecular interaction model of harmine bound to the DYRK2 family kinase was generated by computational docking using X-ray structures. NB tumors from human patients were profiled for DYRK mRNA expression patterns and clinical correlations using the R2 platform.ResultsThe IC50 values for harmine after 72 h treatment were 169.6, 170.8, and 791.7 μM for SKNBE, KELLY, and SKNFI, respectively. Exposure of these NB cell lines to 100 μM of harmine resulted in caspase-3/7 and caspase-9 activation as well as caspase-mediated PARP cleavage and Annexin V-positive stained cells, as early as 24 h after treatment, clearly suggesting apoptosis induction, especially in MYCN-amplified cell lines. Elevated DYRK2 mRNA levels correlated with poor prognosis in a large cohort of NB tumors.ConclusionHarmine is a known inhibitor of DYRK family kinases. It can induce apoptosis in NB cell lines, which led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that DYRK inhibition by harmine and the subsequent triggering of caspase-mediated apoptosis might present a novel approach to NB therapy.

DYRK1A Kinase Positively Regulates Angiogenic Responses in Endothelial Cells

Angiogenesis is a highly regulated process essential for organ development and maintenance, and its deregulation contributes to inflammation, cardiac disorders, and cancer. The Ca2+/nuclear factor of activated Tcells (NFAT) signaling pathway is central to endothelial cell angiogenic responses, and it is activated by stimuli like vascular endothelial growth factor (VEGF) A. NFAT phosphorylation by dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) is thought to be an inactivating event. Contrary to expectations, we show that the DYRK family member DYRK1A positively regulates VEGF-dependent NFAT transcriptional responses in primary endothelial cells. DYRK1A silencing reduces intracellular Ca2+ influx in response to VEGF, which dampens NFAT activation. The effect is exerted at the level of VEGFR2 accumulation leading to impairment in PLCγ1 activation. Notably, Dyrk1a heterozygous mice show defects in developmental retinal vascularization. Our data establish a regulatory circuit, DYRK1A/ Ca2+/NFAT, to fine-tune endothelial cell proliferation and angiogenesis.

Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 μM/SC84227, 0.60 ± 0.02 μM/SC97202, 6.26 ± 1.64 μM/SC97208, 0.71 ± 0.019 μM/Harmine and 0.63 ± 0.23 μM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti-herpesviral strategies.

Structural Optimization and Pharmacological Evaluation of Inhibitors Targeting Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases (DYRK) and CDC-like kinases (CLK) in Glioblastoma.

The DYRK family contains kinases that are up-regulated in malignancy and control several cancer hallmarks. To assess the anticancer potential of inhibitors targeting DYRK kinases, we developed a series of novel DYRK inhibitors based on the 7-azaindole scaffold. All compounds were tested for their ability to inhibit DYRK1A, DYRK1B, DYRK2, and the structurally related CLK1. The library was screened for anticancer efficacy in established and stem cell-like glioblastoma cell lines. The most potent inhibitors (IC50 ≤ 50 nM) significantly decreased viability, clonogenic survival, migration, and invasion of glioblastoma cells. Target engagement was confirmed with genetic knockdown and the cellular thermal shift assay. We demonstrate that DYRK1A's thermal stability in cells is increased upon compound treatment, confirming binding in cells. In summary, we present synthesis, structure-activity relationship, and efficacy in glioblastoma-relevant models for a library of novel 7-azaindoles.

DYRK1A Is a Therapeutic Target in B-ALL in Children with Down Syndrome

Children with Down syndrome have a spectrum of associated disorders including a 20-fold increased incidence of B-cell acute lymphoblastic leukemia (DS-ALL). Although a number of genetic alterations have been found in this ALL subtype, such as activating mutations in JAK2 and overexpression of CRLF2, the mechanisms by which trisomy 21 promotes the leukemia are largely unknown. Previous studies have implicated chromosome 21 genes HMGN1 and DYRK1A in both malignant and normal lymphopoiesis.DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family that has been well studied in non-hematopoietic tissues. Its targets include proteins that regulate multiple pathways including cell signaling, cell cycle, and brain development. We have previously shown that DYRK1A is a megakaryoblastic leukemia-promoting gene through its negative regulation of NFAT transcription factors. Furthermore, in studies with a conditional Dyrk1a knock-out mouse, we found that the kinase is required for lymphoid, but not myeloid cell development. In developing lymphocytes, Dyrk1a regulates the cell cycle by destabilizing cyclin D3. Consequently, loss of Dyrk1a resulted in the failure of these cells to switch from a proliferative to quiescent phase for subsequent maturation (Thompson et al. J. Exp. Med. 2015 212:953-70). Despite this deficiency in exiting the cell cycle, Dyrk1a-deficient lymphocytes also exhibit impaired proliferation before undergoing apoptosis. These data reveal a critical role for DYRK1A in lymphopoiesis and suggest that it may be a target for therapeutic intervention.We assayed the activity of the highly selective and potent DYRK1 inhibitor, EHT 1610, in multiple ALL cell lines. EHT 1610 inhibited the growth of Jurkat and MHH-CALL-4 cells with EC50s of 0.83mM and 0.49mM, respectively. Next, we treated primary human ALL blasts with EHT 1610 and the less selective DYRK1A inhibitor harmine. Growth of 16 out of 30 specimens, which included DS-ALL, pre-B ALL, and T-ALL, was sensitive to DYRK1A inhibition at doses between 0.5 and 10mM. Of note, growth of 9 of the 11 of the DS-ALL samples was inhibited by EHT 1610. This result indicates that the increased dosage of DYRK1A in DS samples sensitizes the cells to DYRK1A inhibition.To further study the contributions of DYRK1A to normal and malignant lymphopoiesis, we performed phosphoproteomic analysis on primary murine pre-B cells treated with EHT 1610. After 2 hours of EHT 1610 treatment, the cells were collected and analyzed for changes in the phosphoproteome. Phosphorylation of 36 proteins was significantly altered. Bioinformatics analysis led to the identification of a number of notable pathways that appear to be regulated by DYRK1A including cell cycle, cell division and mitosis, RNA metabolism, and JAK-STAT signaling. Differentially phosphorylated proteins included geminin, which is important in cell division and whose loss enhances megakaryopoiesis, and POLR2M, which is intriguing because DYRK1A phosphorylates the CTD of RNA Pol II and binds chromatin at specific sites in glioblastoma cells. Another interesting target is STAT3, which is phosphorylated by DYRK1A on Ser727, a residue whose phosphorylation is required for maximal STAT3 activation. Treatment of murine pre-B cells with EHT 1610 significantly reduced the level of phosphorylation of Ser727 and Tyr705, suggesting that DYRK1A may provide a priming event for STAT3 activation similar to its priming effect on GSK3b phosphorylation. Consistent with a role for JAK/STAT signaling and STAT3 activity, B-ALL cells were highly sensitive to ruxolitinib therapy. Taken together, our study suggests that DYRK1A is a therapeutic target in DS-ALL and likely functions in part by enhancing JAK/STAT signaling. DisclosuresNo relevant conflicts of interest to declare.

The DYRK-family kinase Pom1 phosphorylates the F-BAR protein Cdc15 to prevent division at cell poles.

Division site positioning is critical for both symmetric and asymmetric cell divisions. In many organisms, positive and negative signals cooperate to position the contractile actin ring for cytokinesis. In rod-shaped fission yeast Schizosaccharomyces pombe cells, division at midcell is achieved through positive Mid1/anillin-dependent signaling emanating from the central nucleus and negative signals from the dual-specificity tyrosine phosphorylation-regulated kinase family kinase Pom1 at the cell poles. In this study, we show that Pom1 directly phosphorylates the F-BAR protein Cdc15, a central component of the cytokinetic ring. Pom1-dependent phosphorylation blocks Cdc15 binding to paxillin Pxl1 and C2 domain protein Fic1 and enhances Cdc15 dynamics. This promotes ring sliding from cell poles, which prevents septum assembly at the ends of cells with a displaced nucleus or lacking Mid1. Pom1 also slows down ring constriction. These results indicate that a strong negative signal from the Pom1 kinase at cell poles converts Cdc15 to its closed state, destabilizes the actomyosin ring, and thus promotes medial septation.

Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency

Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency.

Splice Variants of the Dual Specificity Tyrosine Phosphorylation-regulated Kinase 4 (DYRK4) Differ in Their Subcellular Localization and Catalytic Activity

Dual specificity tyrosine phosphorylation-regulated kinases, DYRKs, are a family of conserved protein kinases that play key roles in the regulation of cell differentiation, proliferation, and survival. Of the five mammalian DYRKs, DYRK4 is the least studied family member. Here, we show that several splice variants of DYRK4 are expressed in tissue-specific patterns and that these variants have distinct functional capacities. One of these variants contains a nuclear localization signal in its extended N terminus that mediates its interaction with importin α3 and α5 and that is capable of targeting a heterologous protein to the nucleus. Consequently, the nucleocytoplasmic mobility of this variant differs from that of a shorter isoform in live cell imaging experiments. Other splicing events affect the catalytic domain, including a three-amino acid deletion within subdomain XI that markedly reduces the enzymatic activity of DYRK4. We also show that autophosphorylation of a tyrosine residue within the activation loop is necessary for full DYRK4 kinase activity, a defining feature of the DYRK family. Finally, by comparing the phosphorylation of an array of 720 peptides, we show that DYRK1A, DYRK2, and DYRK4 differ in their target recognition sequence and that preference for an arginine residue at position P -3 is a feature of DYRK1A but not of DYRK2 and DYRK4. Therefore, we highlight the use of subcellular localization as an important regulatory mechanism for DYRK proteins, and we propose that substrate specificity could be a source of functional diversity among DYRKs.

Characterization of DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) from Zebrafish (Dario rerio)

Proteins of the DYRK (dual-specificity tyrosine-phosphorylation-regulated kinase) family are characterized by the presence of a conserved kinase domain and N-terminal DH box. DYRK2 is involved in regulating key developmental and cellular processes, such as neurogenesis, cell proliferation, cytokinesis, and cellular differentiation. Herein, we report that the ortholog of DYRK2 found in zebrafish shares about 70% identity with that of human, mouse, and chick. RT-PCR showed that DYRK2 is expressed maternally and zygotically. In-situ hybridization results show that DYRK2 is expressed in somite cells that will develop into muscles. Our results provide preliminary evidence for investigating the in-vivo function of DYRK2 in zebrafish muscle development.

The Characterization of DYRK3 mRNA and the Utility of DYRK3 Inhibitors in a Carboplatin/Radiation Mouse Model of Anemia.

DYRK3, a member of the dual-specificity tyrosine phosphorylation-regulated kinase family, is expressed at low levels in erythroid progenitors and is implicated as a negative regulator of erythropoiesis. An appropriate animal model of anemia was sought to investigate the potential utility of DYRK3 inhibitors as a therapy for the treatment of anemia. Treatment of mice with sub-lethal irradiation followed by a single dose of the chemotherapeutic, nucleoside analog carboplatin, is well known to induce severe anemia. In the first instance, an analysis of DYRK3 mRNA levels was performed to ascertain whether the anemia so induced results in increased DYRK3 transcription. In this carboplatin/radiation model, mice were exposed to sub-lethal gamma irradiation (500 rads) followed by a single dose of carboplatin (60 mg/kg). This treatment induced an approximately 50% decrease in hemoglobin with concomitant drops in other erythroid parameters (hematocrit and RBC) by days 15–17. The following parameters were examined in five animals per day: peripheral blood counts, marrow cell count, plasma Epo levels, marrow Ter119+/CD71+ expression and marrow DYRK3 mRNA. As expected, the hemoglobin, hematocrit and red blood cells decreased gradually to nadir on day 15. White blood cells decreased to very low levels within 2 days of carboplatin/radiation treatment and remained suppressed for 11 days. Platelets decreased to nadir at day 7, where they remain until day 10. Plasma Epo levels were low and abruptly increased at day 3–4. The absolute number of Ter119+/CD71+ cells immediately dropped from normal levels at day 1 and then increased at day 6 and then fluctuated between a 30- to 60- fold enhancement from day 8 through 21 when the study was complete. DYRK3 mRNA, as measured by quantitative PCR (Taqman), increased approximately 10-fold at day 7 and remained in that range until day 21. The number of erythroid progenitors measured by flow cytometry,Ter119+/CD71+ cells, and the level of DYRK3 mRNA remained elevated until the end of the experiment at day 21, at which point the hemoglobin had recovered to near normal levels. GSK626616, a potent, low molecular weight inhibitor of DYRK3 kinase activity (IC50 = 0.7 nM), was dosed once daily, i.p., for 17 days in this model. At day 15, the GSK626616-treated mice (0.03 mg/kg) demonstrated a statistically significant increase in hemoglobin, hematocrit, red blood cell and platelet counts compared to anemic animals treated with vehicle alone. In contrast to its effects in anemic mice, this compound demonstrated no increases in any blood parameters in normal mice over a similar timeframe and dosage regimen. This expected behavior is hypothesized to be due to the low level of DYRK3 mRNA in normal, non-anemic mice. The characterization of this carboplatin/radiation mouse model demonstrates that as the hemoglobin decreased, plasma Epo increased at day 3–4, followed by the increase in Ter119+/CD71+ cells at day 6. Following this surge in erythropoiesis, an increase in DYRK3 mRNA expression naturally follows. The subsequent improved erythropoiesis in animals treated with a DYRK3 inhibitor in this model, suggests that DYRK3 kinase mRNA levels could have utility as a biomarker in the identification of an anemic patient population that then may be a candidate for treatment with a DYRK3 inhibitor.

GSK626616: A DYRK3 Inhibitor as a Potential New Therapy for the Treatment of Anemia.

Through both in vitro and in vivo validation studies, such as antisense RNA and gene knock-out experiments, DYRK3 has been implicated as a negative regulator of erythropoiesis. DYRK3, a member of the dual-specificity tyrosine phosphorylation-regulated kinase family, is expressed at low levels in erythroid progenitors and plays a regulatory role in their cellular proliferation and differentiation. High-throughput screening at GSK resulted in the identification of a novel series of thiazolidinone inhibitors of DYRK3. Lead optimization efforts then led to the discovery of GSK626616 as a potent, orally bioavailable inhibitor of DYRK3. GSK626616, inhibits DYRK3 in vitro with an IC50of 0.7 nM. This low molecular weight compound (401 Da) inhibits other members of the DYRK family, e.g., DYRK1A and DYRK2, with similar potency and with an approximate 20-fold selectivity versus the next most potently inhibited kinase, casein kinase 2. GSK626616AC, the meglumine, monohydrate salt form, has high oral bioavailability in all pre-clinical species studied (e.g. canine AUC = 23.64 ± 2.84 μghr/mL for a 30 mg/kg dose presented as crystalline material packed within a capsule). In cellular assays, GSK626616 enhances the number of CFU-E stimulated by Epo from human marrow, although it has no CFU-E activity on its own, consistent with the target's functional role as a negative regulator. GSK626616 is specific for the erythroid lineage and does not stimulate CFU-GM colonies, either alone or in the presence of G-CSF or GM-CSF. There is also no direct effect on megakaryocyte colony growth from progenitor cells. 3H-thymidine incorporation in kit+ murine marrow is also stimulated after 3 days by exposure to GSK626616 in the presence of SCF and EPO. Similarly, 3 day treatment with GSK626616 increased the percentage, as well as the absolute number, of Ter119+/CD71+ erythroid progenitors derived from kit+ mouse marrow in the presence of SCF and Epo. GSK626616 (0.0001 to 30 uM) dosed i.p., daily for 14 days had no effect on the blood counts of normal mice, either alone or in the presence of 200 or 600 U/kg Epo. This was not unexpected based upon the very low levels of DYRK3 expression in the bone marrow of normal, non-anemic mice and on results previously reported for knock-out mice. These mice have normal erythropoiesis, but demonstrate an enhanced recovery from erythropoietic stress (Wojchowski, Blood 2005). Anemic mice, treated with a daily dose of GSK626616, i.p., had a statistically significant increase in hemoglobin compared to those dosed with vehicle alone at day 15 after anemia was induced through carboplatin/radiation treatment. Platelet levels were also elevated compared to vehicle control at day 15. These data suggest that treatment with GSK626616, through inhibition of DYRK3 activity, leads to an increase in the proliferation of kit+ cells producing an increased number of Ter119+/CD71+ erythroblasts, thereby accelerating recovery from the anemic insult in a carboplatin/radiation mouse model. It is hypothesized that this mechanism may only function under anemic conditions when DYRK3 is elevated and therefore, that the effects will be self-regulated as hemoglobin and EPO levels approach the normal range.