Emerging roles of DYRK2 in cancer.

Over the last decade, the CMGC kinase DYRK2 has been reported as a tumor suppressor across various cancers triggering major antitumor and proapoptotic signals in breast, colon, liver, ovary, brain, and lung cancers, with lower DYRK2 expression correlated with poorer prognosis in patients. Contrary to this, various medicinal chemistry studies reported robust antiproliferative properties of DYRK2 inhibitors, whereas unbiased 'omics' and genome-wide association study-based studies identified DYRK2 as a highly overexpressed kinase in various patient tumor samples. A major paradigm shift occurred in the last 4 years when DYRK2 was found to regulate proteostasis in cancer via a two-pronged mechanism. DYRK2 phosphorylated and activated the 26S proteasome to enhance degradation of misfolded/tumor-suppressor proteins while also promoting the nuclear stability and transcriptional activity of its substrate, heat-shock factor 1 triggering protein folding. Together, DYRK2 regulates proteostasis and promotes protumorigenic survival for specific cancers. Indeed, potent and selective small-molecule inhibitors of DYRK2 exhibit in vitro and in vivo anti-tumor activity in triple-negative breast cancer and myeloma models. However, with conflicting and contradictory reports across different cancers, the overarching role of DYRK2 remains enigmatic. Specific cancer (sub)types coupled to spatiotemporal interactions with substrates could decide the procancer or anticancer role of DYRK2. The current review aims to provide a balanced and critical appreciation of the literature to date, highlighting top substrates such as p53, c-Myc, c-Jun, heat-shock factor 1, proteasome, or NOTCH1, to discuss DYRK2 inhibitors available to the scientific community and to shed light on this duality of protumorigenic and antitumorigenic roles of DYRK2.

Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.

Phosphorylation-dependent regulation of the NOTCH1 intracellular domain by dual-specificity tyrosine-regulated kinase 2.

NOTCH proteins constitute a receptor family with a widely conserved role in cell cycle, growing and development regulation. NOTCH1, the best characterised member of this family, regulates the expression of key genes in cell growth and angiogenesis, playing an essential role in cancer development. These observations provide a relevant rationale to propose the inhibition of the intracellular domain of NOTCH1 (Notch1-IC) as a strategy for treating various types of cancer. Notch1-IC stability is mainly controlled by post-translational modifications. FBXW7 ubiquitin E3 ligase-mediated degradation is considered one of the most relevant, being the previous phosphorylation at Thr-2512 residue required. In the present study, we describe for the first time a new regulation mechanism of the NOTCH1 signalling pathway mediated by DYRK2. We demonstrate that DYRK2 phosphorylates Notch1-IC in response to chemotherapeutic agents and facilitates its proteasomal degradation by FBXW7 ubiquitin ligase through a Thr-2512 phosphorylation-dependent mechanism. We show that DYRK2 regulation by chemotherapeutic agents has a relevant effect on the viability, motility and invasion capacity of cancer cells expressing NOTCH1. In summary, we reveal a novel mechanism of regulation for NOTCH1 which might help us to better understand its role in cancer biology.

A redox-regulated SUMO/acetylation switch of HIPK2 controls the survival threshold to oxidative stress.

Moderate concentrations of reactive oxygen species (ROS) serve as coregulatory signaling molecules, whereas exceedingly high concentrations trigger cell death. Here, we identify ROS-induced acetylation of the proapoptotic kinase HIPK2 as a molecular mechanism that controls the threshold discerning sensitivity from resistance toward ROS-mediated cell death. SUMOylation of HIPK2 at permissive ROS concentrations allows the constitutive association of HDAC3 and keeps HIPK2 in the nonacetylated state. Elevated ROS concentrations prevent SUMOylation of HIPK2 and, consequently, reduce association of HDAC3, thus leading to the acetylation of HIPK2. Reconstitution experiments showed that HIPK2-dependent genes cause decreased ROS levels. Although a nonacetylatable HIPK2 mutant enhanced ROS-induced cell death, an acetylation-mimicking variant ensured cell survival even under conditions of high oxidative stress.

Induction of the Antioxidant Response by the Transcription Factor NRF2 Increases Bioactivation of the Mutagenic Air Pollutant 3-Nitrobenzanthrone in Human Lung Cells.

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen present in diesel exhaust. It requires metabolic activation via nitroreduction in order to form DNA adducts and promote mutagenesis. We have determined that human aldo-keto reductases (AKR1C1-1C3) and NAD(P)H:quinone oxidoreductase 1 (NQO1) contribute equally to the nitroreduction of 3-NBA in lung epithelial cell lines and collectively represent 50% of the nitroreductase activity. The genes encoding these enzymes are induced by the transcription factor NF-E2 p45-related factor 2 (NRF2), which raises the possibility that NRF2 activation exacerbates 3-NBA toxification. Since A549 cells possess constitutively active NRF2, we examined the effect of heterozygous (NRF2-Het) and homozygous NRF2 knockout (NRF2-KO) by CRISPR-Cas9 gene editing on the activation of 3-NBA. To evaluate whether NRF2-mediated gene induction increases 3-NBA activation, we examined the effects of NRF2 activators in immortalized human bronchial epithelial cells (HBEC3-KT). Changes in AKR1C1-1C3 and NQO1 expression by NRF2 knockout or use of NRF2 activators were confirmed by qPCR, immunoblots, and enzyme activity assays. We observed decreases in 3-NBA activation in the A549 NRF2 KO cell lines (53% reduction in A549 NRF2-Het cells and 82% reduction in A549 NRF2-KO cells) and 40-60% increases in 3-NBA bioactivation due to NRF2 activators in HBEC3-KT cells. Together, our data suggest that activation of the transcription factor NRF2 exacerbates carcinogen metabolism following exposure to diesel exhaust which may lead to an increase in 3-NBA-derived DNA adducts.

Homeodomain-interacting protein kinase 2-dependent repression of myogenic differentiation is relieved by its caspase-mediated cleavage.

Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.

Octyl itaconate enhances VSVΔ51 oncolytic virotherapy by multitarget inhibition of antiviral and inflammatory pathways.

The presence of heterogeneity in responses to oncolytic virotherapy poses a barrier to clinical effectiveness, as resistance to this treatment can occur through the inhibition of viral spread within the tumor, potentially leading to treatment failures. Here we show that 4-octyl itaconate (4-OI), a chemical derivative of the Krebs cycle-derived metabolite itaconate, enhances oncolytic virotherapy with VSVΔ51 in various models including human and murine resistant cancer cell lines, three-dimensional (3D) patient-derived colon tumoroids and organotypic brain tumor slices. Furthermore, 4-OI in combination with VSVΔ51 improves therapeutic outcomes in a resistant murine colon tumor model. Mechanistically, we find that 4-OI suppresses antiviral immunity in cancer cells through the modification of cysteine residues in MAVS and IKKß independently of the NRF2/KEAP1 axis. We propose that the combination of a metabolite-derived drug with an oncolytic virus agent can greatly improve anticancer therapeutic outcomes by direct interference with the type I IFN and NF-κB-mediated antiviral responses.

Nrf2 depletion in the context of loss-of-function Keap1 leads to mitolysosome accumulation.

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) is the principal determinant of the cellular redox homeostasis, contributing to mitochondrial function, integrity and bioenergetics. The main negative regulator of Nrf2 is Kelch-like ECH associated protein 1 (Keap1), a substrate adaptor for Cul3/Rbx1 ubiquitin ligase, which continuously targets Nrf2 for ubiquitination and proteasomal degradation. Loss-of-function mutations in Keap1 occur frequently in lung cancer, leading to constitutive Nrf2 activation. We used the human lung cancer cell line A549 and its CRISPR/Cas9-generated homozygous Nrf2-knockout (Nrf2-KO) counterpart to assess the role of Nrf2 on mitochondrial health. To confirm that the observed effects of Nrf2 deficiency are not due to clonal selection or long-term adaptation to the absence of Nrf2, we also depleted Nrf2 by siRNA (siNFE2L2), thus creating populations of Nrf2-knockdown (Nrf2-KD) A549 cells. Nrf2 deficiency decreased mitochondrial respiration, but increased the mitochondrial membrane potential, mass, DNA content, and the number of mitolysosomes. The proportion of ATG7 and ATG3 within their respective LC3B conjugates was increased in Nrf2-deficient cells with mutant Keap1, whereas the formation of new autophagosomes was not affected. Thus, in lung cancer cells with loss-of-function Keap1, Nrf2 facilitates mitolysosome degradation thereby ensuring timely clearance of damaged mitochondria.

Dual inhibition of HSF1 and DYRK2 impedes cancer progression.

Preserving proteostasis is a major survival mechanism for cancer. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a key oncogenic kinase that directly activates the transcription factor heat-shock factor 1 (HSF1) and the 26S proteasome. Targeting DYRK2 has proven to be a tractable strategy to target cancers sensitive to proteotoxic stress; however, the development of HSF1 inhibitors remains in its infancy. Importantly, multiple other kinases have been shown to redundantly activate HSF1 that promoted ideas to directly target HSF1. The eventual development of direct HSF1 inhibitor KRIBB11 suggests that the transcription factor is indeed a druggable target. The current study establishes that concurrent targeting of HSF1 and DYRK2 can indeed impede cancer by inducing apoptosis faster than individual targetting. Furthermore, targeting the DYRK2-HSF1 axis induces death in proteasome inhibitor-resistant cells and reduces triple-negative breast cancer (TNBC) burden in ectopic and orthotopic xenograft models. Together the data indicate that cotargeting of kinase DYRK2 and its substrate HSF1 could prove to be a beneficial strategy in perturbing neoplastic malignancies.

Control of nuclear HIPK2 localization and function by a SUMO interaction motif.

The serine/threonine kinase HIPK2 regulates gene expression programs controlling differentiation and cell death. HIPK2 localizes in subnuclear speckles, but the structural components allowing this localization are not understood. A point mutation analysis allowed mapping two nuclear localization signals and a SUMO interaction motif (SIM) that also occurs in HIPK1 and HIPK3. The SIM binds all three major isoforms of SUMO (SUMO-1-3), while only SUMO-1 is capable of covalent conjugation to HIPK2. Deletion or mutation of the SIM prevented SUMO binding and precluded localization of HIPK2 in nuclear speckles, thus causing localization of HIPK2 to the entire cell. Functional inactivation of the SIM prohibited recruitment of HIPK2 to PML nuclear bodies and disrupted colocalization with other proteins such as the polycomb protein Pc2 in nuclear speckles. Interaction of HIPK2 with Pc2 or PML in intact cells was largely dependent on a functional SIM in HIPK2, highlighting the relevance of SUMO/SIM interactions as a molecular glue that serves to enhance protein/protein interaction networks. HIPK2 mutants with an inactive SIM showed changed activities, thus revealing that non-covalent binding of SUMO to the kinase is important for the regulation of its function.