Cyclin-dependent Kinase Phosphorylation Research Articles

Abstract 1985: Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells

Abstract Approximately 75% of breast tumors express estrogen receptors (ER+); recurrent ER+ breast cancer is generally incurable. Endocrine therapy (ET) is the mainstay of treatment for ER+ breast cancer and works by targeting either ER activity (Tamoxifen or Fulvestrant) or availability of its ligand estrogen (Letrozole, Exemestane). Unfortunately, endocrine therapies are limited by the emergence of resistance; resistance to endocrine therapies (ET) remains a significant clinical challenge. We have explored the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway, in ET-resistant breast cancer cells. Protein expression of ADSL was significantly higher in two different ET-resistant cells (LCC9 and T47D-4HT) when compared with their respective parental ET-sensitive cells (MCF-7 and T47D). siRNA mediated ablation of ADSL in LCC9 and T47D-4HT cells blocked cell proliferation and both colony and spheroid formation. Depletion of ADSL in LCC9 cells obstructed cell cycle progression from G1 to S phase and elevated the protein expression of cyclin D1/D3, CDK2/4, and cyclin E. In T47D-4HT cells, ADSL depletion led to an accumulation of cells in S-phase with reduced protein expression of cyclin D1. Notably, ADSL ablation in LCC9 cells lead to DNA replication stress, resulting in activation (phosphorylation) of ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its key downstream effector checkpoint kinase 1 (Chk1), which further limited the phosphatase activity of cdc25A leading to deactivation (phosphorylation) of Cyclin-Dependent Kinase 2 (CDK2). ADSL knockdown reduced the mitochondrial membrane potential and impaired the oxygen consumption (OCR) and extracellular acidification (ECAR) rates in both LCC9 and T47D-4HT cells. Of note, addition of AICAR to ADSL-depleted LCC9 cells significantly increased cell proliferation, colony formation, and restored mitochondrial membrane potential. RNA sequencing data revealed the effect of ADSL depletion on enrichment of different pathways in LCC9 and TH47D-4HT cells. Importantly, p53 signaling was significantly enriched in LCC9 cells but not in T47D-4HT cells after ADSL knockdown (T47D cells have a mutated p53 gene). ER+ breast cancer patients treated with ET showed high ADSL expression that is associated with poor recurrence-free survival. In summary, our findings indicate that ADSL-mediated de novo purine synthesis is crucial for cell proliferation. Therefore, targeting ADSL may be a novel therapeutic approach for ER, ET-resistant breast cancer. Citation Format: ANIL KUMAR Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1985.

The role of RNF138 in DNA end resection is regulated by ubiquitylation and CDK phosphorylation

Double-strand breaks (DSBs) are DNA lesions that pose a significant threat to genomic stability. The repair of DSBs by the homologous recombination (HR) pathway is preceded by DNA end resection, the 5' to 3' nucleolytic degradation of DNA away from the DSB. We and others previously identified a role for RNF138, a really interesting new gene finger E3 ubiquitin ligase, in stimulating DNA end resection and HR. Yet, little is known about how RNF138's function is regulated in the context of DSB repair. Here, we show that RNF138 is phosphorylated at residue T27 by cyclin-dependent kinase (CDK) activity during the S and G2 phases of the cell cycle. We also observe that RNF138 is ubiquitylated constitutively, with ubiquitylation occurring in part on residue K158 and rising during the S/G2 phases. Interestingly, RNF138 ubiquitylation decreases upon genotoxic stress. By mutating RNF138 at residues T27, K158, and the previously identified S124 ataxia telangiectasia mutated phosphorylation site (Han etal., 2016, ref. 22), we find that post-translational modifications at all three positions mediate DSB repair. Cells expressing the T27A, K158R, and S124A variants of RNF138 are impaired in DNA end resection, HR activity, and are more sensitive to ionizing radiation compared to those expressing wildtype RNF138. Our findings shed more light on how RNF138 activity is controlled by the cell during HR.

CDK signaling via nonconventional CDK phosphorylation sites.

Since the discovery of cyclin-dependent kinases (CDKs), it has been perceived as a dogma that CDK signaling in the cell cycle is mediated via targeting the CDK consensus sites: the optimal and the minimal motifs S/T-P-x-K/R and S/T-P, respectively. However, more recent evidence suggests that often the CDK phosphorylation events of regulatory importance are mediated via nonconventional CDK sites that lack the required +1Pro of the consensus site motif. In these cases, the loss of specificity seems to be compensated via distant docking interactions facilitated by 1) phosphorylated priming sites binding to phospho-adaptor Cks1 and/or 2) cyclin-specific docking interactions via Short Linear Motifs (SLiMs) in substrates. This Perspective discusses the possible reasons why nonconventional CDK sites are used for CDK signaling. First, the nonconventional CDK sites can act as specificity filters to recognize and distinguish the CDK signal from many other proline-directed kinases in cells. Second, the nonconventional CDK sites in combination with the docking mechanisms provide a much wider range of phosphorylation rates, and thus, also a wider range of CDK thresholds during the accumulation and decline of CDK activity during the cell cycle. As a large number of Cks1-dependent nonconventional CDK sites have been discovered recently, past studies focusing on mutating only the consensus sites should likely be critically reexamined. It is also very likely that phosphorylation of nonconventional sites is crucial in many other kinase-signaling networks.

Delineation of the CENP-LN sub-complex dissociation mechanism upon multisite phosphorylation during mitosis

Phosphorylation is the most prevalent form of regulation in cells, organizing virtually all cellular functions, including survival, motility, differentiation, proliferation, and metabolism. This regulatory function has been largely conserved from the primitive single-cell to the more complex multicellular organisms. More than a third of proteins in eukaryotes are phosphorylated, and essentially every class of protein undergoes regulation by phosphorylation. A decline in the cellular level of CENP-L and CENP-N (components of the constitutive centromere associated network) has earlier been reported and linked to cyclin-dependent kinase (CDK) phosphorylation upon transition into mitosis. Given the importance of posttranslational modifications in cell cycle regulation, mechanistic comprehension of the impact of phosphorylation on both proteins (CENP-L and CENP-N) is of high significance. Through the application of diverse computational analytical techniques, including atomistic molecular dynamics simulations, the mechanism of kinetochore mis-localization and dissociation of the CENP-LN sub-complex in mitosis was delineated. We showed that the phosphorylation of both components of the sub-complex induces global conformational destabilizing effects on the proteins, combined with changes in the electrostatic potential and increase in steric clashes around the protein-protein interaction interface. This, consistent with earlier experimental reports, suggest that the multisite phosphorylation of the CENP-LN sub-complex plays a crucial role in the regulation of cell division. Communicated by Ramaswamy H. Sarma

Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 is well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. The phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO complex formation and we provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO complex formation.

Aconitate decarboxylase 1 is a mediator of polymicrobial sepsis.

Sepsis is a challenging clinical syndrome caused by a dysregulated host response to infection. Here, we identified an unexpected proseptic activity of aconitate decarboxylase 1 (ACOD1) in monocytes and macrophages. Previous studies have suggested that ACOD1, also known as immune-responsive gene 1, is an immunometabolic regulator that favors itaconate production to inhibit bacterial lipopolysaccharide-induced innate immunity. We used next-generation sequencing of lipopolysaccharide-activated THP1 cells to demonstrate that ACOD1 accumulation confers a robust proinflammation response by activating a cytokine storm, predominantly through the tumor necrosis factor signaling pathway. We further revealed that the phosphorylation of cyclin-dependent kinase 2 (CDK2) on threonine-160 mediates the activation of mitogen-activated protein kinase 8 through receptor for activated C kinase 1, leading to JUN-dependent transcription of ACOD1 in human and mouse macrophages or monocytes. Genetic deletion of CDK2 or ACOD1 in myeloid cells, or the administration of the CDK inhibitor dinaciclib, protected mice against polymicrobial sepsis and was associated with improved survival and decreased cytokine storm. The expression of the CDK2-ACOD1 axis also correlated with severity of illness in a cohort of 40 patients with bacterial sepsis. Thus, our findings provide evidence for a previously unrecognized function of ACOD1 in innate immunity and suggest it as a potential therapeutic target for the treatment of sepsis.

Dihydroartemisinin regulates immune cell heterogeneity by triggering a cascade reaction of CDK and MAPK phosphorylation

Artemisinin (ART) and dihydroartemisinin (DHA), apart from their profound anti-malaria effect, can also beneficially modulate the host immune system; however, the underlying molecular mechanisms remain unclear. Here, we report that DHA selectively induced T-cell activation, with an increased proportion of Ki67+CD4+ T cells, CD25+CD4+ T cells, interferon (IFN)-γ-producing CD8+ T cells, Brdu+ CD8+ T cells and neutrophils, which was found to enhance cellular immunity to experimental malaria and overcome immunosuppression in mice. We further revealed that DHA upregulated the expression of cell proliferation-associated proteins by promoting the phosphorylation of mitogen-activated protein kinase (MAPK), cyclin-dependent kinases (CDKs), and activator protein 1 in the spleen. This study is the first to provide robust evidence that DHA selectively induced the expansion of subsets of splenic T cells through phosphorylated CDKs and MAPK to enhance cellular immune responses under non-pathological or pathological conditions. The data significantly deepened our knowledge in the mechanism underlying DHA-mediated immunomodulation.

Intra-S phase checkpoint kinase Chk1 dissociates replication proteins Treslin and TopBP1 through multiple mechanisms during replication stress

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin–TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin–TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin–TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.

A mechanism of origin licensing control through autoinhibition of S. cerevisiae ORC\xb7DNA\xb7Cdc6

The coordinated action of multiple replicative helicase loading factors is needed for the licensing of replication origins prior to DNA replication. Binding of the Origin Recognition Complex (ORC) to DNA initiates the ATP-dependent recruitment of Cdc6, Cdt1 and Mcm2-7 loading, but the structural details for timely ATPase site regulation and for how loading can be impeded by inhibitory signals, such as cyclin-dependent kinase phosphorylation, are unknown. Using cryo-electron microscopy, we have determined several structures of S. cerevisiae ORC·DNA·Cdc6 intermediates at 2.5–2.7 Å resolution. These structures reveal distinct ring conformations of the initiator·co-loader assembly and inactive ATPase site configurations for ORC and Cdc6. The Orc6 N-terminal domain laterally engages the ORC·Cdc6 ring in a manner that is incompatible with productive Mcm2-7 docking, while deletion of this Orc6 region alleviates the CDK-mediated inhibition of Mcm7 recruitment. Our findings support a model in which Orc6 promotes the assembly of an autoinhibited ORC·DNA·Cdc6 intermediate to block origin licensing in response to CDK phosphorylation and to avert DNA re-replication.

Structural conservation of WEE1 and its role in cell cycle regulation in plants

The WEE1 kinase is ubiquitous in plant development and negatively regulates the cell cycle through phosphorylations. However, analogies with the control of the human cell cycle by tyrosine- (Tyr-) phosphorylation of cyclin-dependent kinases (CDKs) are sometimes questioned. In this in silico study, we assessed the structural conservation of the WEE1 protein in the plant kingdom with a particular focus on agronomically valuable plants, the legume crops. We analyzed the phylogenetic distribution of amino-acid sequences among a large number of plants by Bayesian analysis that highlighted the general conservation of WEE1 proteins. A detailed sequence analysis confirmed the catalytic potential of WEE1 proteins in plants. However, some substitutions of an arginine and a glutamate at the entrance of the catalytic pocket, illustrated by 3D structure predictions, challenged the specificity of this protein toward the substrate and Tyr-phosphorylation compared to the human WEE1. The structural differences, which could be responsible for the loss of specificity between human and plants, are highlighted and suggest the involvement of plant WEE1 in more cell regulation processes.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

562TiP A phase Ib dose-escalation study of ZN-c3, a WEE1 inhibitor, in combination with chemotherapy in patients with platinum-resistant or -refractory ovarian, peritoneal, or fallopian tube cancer

Ovarian, peritoneal, and fallopian tube cancer are deadly diseases with similar pathological and clinical features. Despite initial therapy with cytoreductive surgery and platinum-based therapy, many patients experience disease relapse either during (refractory) or within 6 months after (resistant) first-line platinum-based therapy. In these patients, various chemotherapies offer clinical activity but none is universally preferred. WEE1 tyrosine kinase is a critical component of G2/M cell cycle checkpoint control and mediates cell cycle arrest by regulating the phosphorylation of cyclin-dependent kinase 1 (CDK1). Inhibition of WEE1 leads to premature mitotic entry, DNA damage and apoptosis induction, and cell death. ZN-c3 is a novel, selective, and orally bioavailable WEE1 inhibitor that has demonstrated significant antitumor activity in nonclinical in vitro and in vivo models. A first-in-human study identified the maximum tolerated dose (MTD) of ZN-c3 when given as monotherapy. Combining ZN-c3 with chemotherapy may inhibit repair of chemotherapy-induced DNA damage and provide therapeutic benefit in this population. This phase 1b, open-label, multicenter study is evaluating the safety, pharmacokinetics, pharmacodynamics, and anti-tumor activity of ZN-c3 in combination with pegylated liposomal doxorubicin, carboplatin, paclitaxel, or gemcitabine. The primary objectives are to determine MTDs and recommended phase 2 doses for each combination by a modified Bayesian continual reassessment method. Subjects are adult women with high-grade serous epithelial ovarian, peritoneal, or fallopian tube carcinoma who have received 1 or 2 prior chemotherapy regimens including platinum-based therapy and are refractory or resistant to platinum-based therapy. Study drug therapy is given in repeated 21- or 28-day cycles until disease progression or unacceptable toxicity. ZN-c3 is taken orally once daily. Chemotherapy is administered per protocol. Pharmacodynamic effects of therapy in tumor tissue and hair follicle samples will be explored. Subject recruitment is ongoing. NCT04516447 Zentalis Pharmaceuticals.

Activation of PKM2 metabolically controls fulminant liver injury via restoration of pyruvate and reactivation of CDK1

Accumulating evidence indicates that metabolic events profoundly modulate the progression of various diseases. Pyruvate is a central metabolic intermediate in glucose metabolism. In the present study, the metabolic status of pyruvate and its pharmacological significance has been investigated in mice with lipopolysaccharide/D-galactosamine (LPS/D-Gal)-induced fulminant liver injury. Our results indicated that LPS/D-Gal exposure decreased the activity of pyruvate kinase and the content of pyruvate, which were reversed by the PKM2 activator TEPP-46. Pretreatment with TEPP-46 or supplementation with the cell-permeable pyruvate derivate ethyl pyruvate (EP) attenuated LPS/D-Gal-induced liver damage. Interestingly, post-insult intervention of pyruvate metabolism also resulted in beneficial outcomes. The phospho-antibody microarray analysis and immunoblot analysis found that the inhibitory phosphorylation of cyclin dependent kinase 1 (CDK1) was reversed by TEPP-46, DASA-58 or EP. In addition, the therapeutic benefits of PKM2 activator or EP were blunted by the CDK1 inhibitor Ro 3306. Our data suggests that LPS/D-Gal exposure-induced decline of pyruvate might be a novel metabolic mechanism underlies the development of LPS/D-Gal-induced fulminant liver injury, PKM2 activator or pyruvate derivate might have potential value for the pharmacological intervention of fulminant liver injury.

Abstract 1052: 6-phosphofructo-2-kinase enhances cytotoxicity of the EGFR inhibitor erlotinib via regulation of cell cycle in non-small lung cancer cell lines

Abstract Lung tumor development and aggressiveness is largely driven by a multitude of oncogenes including the epidermal growth factor receptor (EGFR). Targeted EGFR inhibition with small-molecule tyrosine kinase inhibitors (EGFR-TKIs) such as erlotinib have initially improved progression-free survival in patients with EGFR-driven NSCLCs but failed to provide an overall survival benefit due to the development of resistance. Clinically, responses to EGFR-TKIs are evaluated by positron emission tomography using 2[18F]- fluoro-2-deoxy-glucose (18FDG-PET) as tumors consume significantly more glucose compared to adjacent normal tissue in vivo. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), a known driver of glycolytic flux, plays a key role in the metabolic shift that occurs during tumor progression. The product of PFKFB3, F2,6BP, regulates glucose metabolism by activating PFK1, which in turn facilitates glycolysis. Increased glycolysis fulfills the elevated tumor demands for energetic and biosynthetic precursors needed for survival and proliferation - the functions that confer resistance to targeted therapies. Here, we interrogated the hypothesis that PFKFB3 inhibition increases the efficacy of EGFR-TKIs in NSCLCs with different EGFR mutation status in vitro. We treated multiple NSCLCs models (wild type EGFR: H522, H1437; and mutated EGFR: PC9, HCC827) with erlotinib and observed a significant inhibition of cell proliferation in vitro. Erlotinib treatment resulted in G1 arrest, decreased expression and phosphorylation of cyclin dependent kinases (CDKs), Rb and accumulation of p27 in all NSCLCs. Notably, PFKFB3 silencing in TKI-treated cells dramatically decreased cell viability, indicating that PFKFB3 expression is required to maintain cell viability of G1 arrested cells. Further, co-treatment with the PFKFB3 inhibitor PFK-158 increased cell death in all NSCLCs. We found that inhibition of PFKFB3 reduced TKI-induced G1 arrest and altered cell cycle progression, switching the effect of erlotinib from cytostatic to cytotoxic. Importantly, dual treatment caused only minor changes in glycolytic flux when compared to the effects of PFK-158 or erlotinib alone suggesting that glucose metabolism did not contribute to the observed effect. Taken together, our findings suggest that a non-metabolic function of PFKFB3 limits the usefulness of EGFR-TKI in vitro and that targeting of PFKFB3 can be a promising approach to improve clinical efficacy of FDA-approved EGFR-TKIs in lung cancer patients. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Jason Chesney, Yoannis Imbert-Fernandez. 6-phosphofructo-2-kinase enhances cytotoxicity of the EGFR inhibitor erlotinib via regulation of cell cycle in non-small lung cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1052.

Accumulation of Nuclear PKM2 Promotes LPS/D-Gal-Induced Fulminant Liver Injury via Reduction of Pyruvate and Inactivation of CDK1

Background: Accumulating evidence indicates that metabolic events profoundly modulate the progression of various diseases. The pyruvate kinases catalyze the production of pyruvate and pyruvate kinase M2 (PKM2) is a unique isoform involved in the development of tumors. In the present study, the pathological significance of PKM2 has been investigated in mice with lipopolysaccharide/D-galactosamine (LPS/D-Gal)-induced fulminant liver injury. Methods: Fulminant liver injury was induced in mice by intraperitoneal injection of LPS/D-Gal, the level of nuclear PKM2, the activity of pyruvate kinase and the content of pyruvate were determined. To investigated the significance of PKM2/ pyruvate, the PKM2 modulator TEPP-46, DASA-58 or the cell permeable ethyl pyruvate (EP) was administered and the degree of hepatocyte apoptosis and liver damage were evaluated. In addition, phospho-antibody microarray was used to identify the underlying signaling pathways. Findings: LPS/D-Gal exposure increased the nuclear level of PKM2 but decreased the activity of pyruvate kinase and the content of pyruvate. Treatment with the PKM2 modulator TEPP-46 or supplementation with ethyl pyruvate (EP) suppressed LPS/D-Gal-induced liver damage. Interestingly, post-insult administration of TEPP-46, another PKM2 modulator DASA-58 or EP also resulted in beneficial outcomes. The phospho-antibody microarray analysis found that LPS/D-Gal induced inhibitory phosphorylation of cyclin dependent kinase 1 (CDK1), which was reversed by TEPP-46, DASA-58 or EP. In addition, the therapeutic benefits of TEPP-46 or EP were blunted by the CDK1 inhibitor Ro 3306. Interpretation: Our data suggests that LPS/D-Gal exposure-induced accumulation of nuclear PKM2, decline of pyruvate and inactivation of CDK1 might be a novel metabolic mechanism underlies the development of LPS/D-Gal-induced fulminant liver injury, PKM2/pyruvate might be a promising target for the pharmacological intervention of fulminant hepatocyte damage. Funding: This work was supported by the grants from the National Natural Science Foundation of China (No. 81871606) and the grant from Science and Technology Research Program of Chongqing Municipal Education Commission (No. KJQN201900435). Declaration of Interest: None to declare. Ethical Approval: The Ethics Committee of Chongqing Medical University approved all mouse experiments.

Mambalgin-2 Induces Cell Cycle Arrest and Apoptosis in Glioma Cells via Interaction with ASIC1a.

Gliomas are fast growing and highly invasive brain tumors, characterized by tumor microenvironment acidification that drives glioma cell growth and migration. Channels containing Acid-sensing Ion Channel 1a subunit (ASIC1a) mediate amiloride-sensitive cation influx in late stage glioma cells, but not in normal astrocytes. Thus, selective targeting of ASIC1a can be a perspective strategy for glioma treatment. Here, ASIC1a expression in U251 MG and A172 glioma cells, but not in normal astrocytes, was demonstrated. Recombinant analog of mambalgin-2 from black mamba Dendroaspis polylepis inhibited amiloride-sensitive currents at ASIC1a both in Xenopus laevis oocytes and in U251 MG cells, while its mutants with impaired activity towards this channel did not. Mambalgin-2 inhibited U251 MG and A172 glioma cells growth with EC50 in the nanomolar range without affecting the proliferation of normal astrocytes. Notably, mambalgin-2 mutants did not affect glioma cell proliferation, pointing on ASIC1a as the main molecular target of mambalgin-2 in U251 MG and A172 cells. Mambalgin-2 induced a cell cycle arrest, inhibited Cyclin D1 and cyclin-dependent kinases (CDK) phosphorylation and caused apoptosis in U251 MG and A172 cells. Moreover, mambalgin-2 inhibited the growth of low-passage primary cells from a patient with glioblastoma. Altogether, our data point to mambalgin-2 as a useful hit for the development of new drugs for glioma treatment.

The Hydrophobic Patch Directs Cyclin B to Centrosomes to Promote Global CDK Phosphorylation at Mitosis.

SummaryThe cyclin-dependent kinases (CDKs) are the major cell-cycle regulators that phosphorylate hundreds of substrates, controlling the onset of S phase and M phase [1, 2, 3]. However, the patterns of substrate phosphorylation increase are not uniform, as different substrates become phosphorylated at different times as cells proceed through the cell cycle [4, 5]. In fission yeast, the correct ordering of CDK substrate phosphorylation can be established by the activity of a single mitotic cyclin-CDK complex [6, 7]. Here, we investigate the substrate-docking region, the hydrophobic patch, on the fission yeast mitotic cyclin Cdc13 as a potential mechanism to correctly order CDK substrate phosphorylation. We show that the hydrophobic patch targets Cdc13 to the yeast centrosome equivalent, the spindle pole body (SPB), and disruption of this motif prevents both centrosomal localization of Cdc13 and the onset of mitosis but does not prevent S phase. CDK phosphorylation in mitosis is compromised for approximately half of all mitotic CDK substrates, with substrates affected generally being those that require the highest levels of CDK activity to become phosphorylated and those that are located at the SPB. Our experiments suggest that the hydrophobic patch of mitotic cyclins contributes to CDK substrate selection by directing the localization of Cdc13-CDK to centrosomes and that this localization of CDK contributes to the CDK substrate phosphorylation necessary to ensure proper entry into mitosis. Finally, we show that mutation of the hydrophobic patch prevents cyclin B1 localization to centrosomes in human cells, suggesting that this mechanism of cyclin-CDK spatial regulation may be conserved across eukaryotes.

The AHI-1-BCR-ABL-DNM2 Complex Mediates Mitochondrial Dynamics in Drug-Resistant BCR-ABL+ Cells

Chronic myeloid leukemia (CML) is driven by the BCR-ABL1 oncoprotein with constitutively active protein-tyrosine kinase activity, perturbing multiple signaling pathways. Although therapies with tyrosine kinase inhibitors (TKIs) can effectively treat early phase CML, relapses and emergence of TKI resistance are problematic, due to BCR-ABL kinase domain mutations and TKI unresponsive quiescent leukemic stem cells (LSCs). These observations point towards a need for alternate treatment strategies to prevent the development of resistant LSCs. We previously demonstrated that Abelson helper integration site-1 (AHI-1) is a highly deregulated protein in CML LSCs and that its WD40-repeat domain physically interacts with BCR-ABL, enhancing leukemia-initiating activity. AHI-1 also contains an SH3 domain, which mediates TKI resistance in LSCs. This domain interacts with dynamin-2 (DNM2) and forms a complex with BCR-ABL, to enhance the phosphorylation and activity of DNM2. The AHI-1-BCR-ABL-DNM2 complex is shown to regulate leukemic properties in patient LSCs, including increased ROS production, endocytosis and autophagy. Interestingly, deletion of the Ahi-1 SH3 domain (Ahi-1 SH3Δ) results in a defect in Ahi-1 localization, with most being present in the nucleus. To test whether Ahi-1 SH3 domain activity directly affects cytoplasmic anchoring and localization, we have generated two Ahi-1 mutants, using site-directed mutagenesis: a mutation in the key tryptophan residue (W939A) involved in SH3 domain binding and in a non-conserved surface residue (M906A), as a negative control, based on the crystal structure of the AHI-1 SH3 domain. Interestingly, the cytoplasm-to-nucleus signal ratio of Ahi-1 W939A was significantly reduced compared to the negative control or wildtype Ahi-1, as assessed by immunofluorescence and confocal microscopy (70% reduction, p<0.0001), indicating that changes in localization of Ahi-1 SH3Δ may result in disruption of the complex and allow for new interactions with nuclear proteins. Investigating changes in the proteome may help uncover downstream effects of the AHI-1-BCR-ABL-DNM2 complex and its biological role in mediating TKI resistance. Advanced antibody microarray analysis was then used to investigate differences in the proteome and phosphorylation landscape of BCR-ABL+ cells co-transduced with wildtype Ahi-1 or Ahi-1 SH3Δ. This system quantifies the differences in expression and phosphorylation states of key signaling proteins simultaneously, using 878 antibodies in duplicate. Twenty leads were identified by the following criteria: a large signal difference of at least 1.5-fold change, high signal strength for high expression, and low error between duplicates. These leads were validated by Western blot analysis and several of them were confirmed. Particularly, phosphorylation of cyclin-dependent kinase 1 (CDK1), a key player in cell cycle control and mitochondrial dynamics, was greatly reduced in cells expressing wildtype Ahi-1 compared to Ahi-1 SH3Δ, indicating that AHI-1-mediated phosphorylation changes in CDK1 may contribute to regulation of mitochondrial functions. Indeed, BCR-ABL-transduced cells co-expressing wildtype Ahi-1 showed increased mitochondria potential in response to TKI treatment or serum starvation, in MitoTracker analysis (p<0.05). However, this was not observed in BCR-ABL-transduced cells co-expressing the Ahi-1 SH3Δ mutant. A similar trend was also observed in immunofluorescence confocal microscopy analysis of the mitochondrial importer receptor, TOM20. To further study the role of DNM2 in mediating mitochondrial dynamics associated with AHI-1 and BCR-ABL, CRISPR-Cas9 mediated DNM2 knockdown was performed in TKI-resistant cells, using two different DNM2-targeting guide RNAs; these resulted in significant reduction in DNM2 (78% & 75%) in Western blot analysis. The knockdown cells showed a reduction in viability (60% reduction) and increased sensitivity to TKI treatment compared to the control (90% vs. 30% reduction) after 48 hours and changes in mitochondrial activity were also observed in these cells. These results support a role for mitochondrial dynamics in the AHI-1-BCR-ABL-DNM2 complex-mediated TKI response and that targeting key biological processes regulated by the AHI-1-BCR-ABL-DNM2 complex and its pathways may lead to new therapeutic strategies to overcome TKI resistance in CML. Disclosures Pelech: Kinexus Bioinformatics Corporation: Equity Ownership.

RecQL4 tethering on the pre-replicative complex induces unscheduled origin activation and replication stress in human cells

Sequential activation of DNA replication origins is precisely programmed and critical to maintaining genome stability. RecQL4, a member of the conserved RecQ family of helicases, plays an essential role in the initiation of DNA replication in mammalian cells. Here, we showed that RecQL4 protein tethered on the pre-replicative complex (pre-RC) induces early activation of late replicating origins during S phase. Tethering of RecQL4 or its N terminus on pre-RCs via fusion with Orc4 protein resulted in the recruitment of essential initiation factors, such as Mcm10, And-1, Cdc45, and GINS, increasing nascent DNA synthesis in late replicating origins during early S phase. In this origin activation process, tethered RecQL4 was able to recruit Cdc45 even in the absence of cyclin-dependent kinase (CDK) activity, whereas CDK phosphorylation of RecQL4 N terminus was required for interaction with and origin recruitment of And-1 and GINS. In addition, forced activation of replication origins by RecQL4 tethering resulted in increased replication stress and the accumulation of ssDNAs, which can be recovered by transcription inhibition. Collectively, these results suggest that recruitment of RecQL4 to replication origins is an important step for temporal activation of replication origins during S phase. Further, perturbation of replication timing control by unscheduled origin activation significantly induces replication stress, which is mostly caused by transcription-replication conflicts.

YWHA (14-3-3) protein isoforms and their interactions with CDC25B phosphatase in mouse oogenesis and oocyte maturation

BackgroundImmature mammalian oocytes are held arrested at prophase I of meiosis by an inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1). Release from this meiotic arrest and germinal vesicle breakdown is dependent on dephosphorylation of CDK1 by the protein, cell cycle division 25B (CDC25B). Evidence suggests that phosphorylated CDC25B is bound to YWHA (14-3-3) proteins in the cytoplasm of immature oocytes and is thus maintained in an inactive form. The importance of YWHA in meiosis demands additional studies.ResultsMessenger RNA for multiple isoforms of the YWHA protein family was detected in mouse oocytes and eggs. All seven mammalian YWHA isoforms previously reported to be expressed in mouse oocytes, were found to interact with CDC25B as evidenced by in situ proximity ligation assays. Interaction of YWHAH with CDC25B was indicated by Förster Resonance Energy Transfer (FRET) microscopy. Intracytoplasmic microinjection of oocytes with R18, a known, synthetic, non-isoform-specific, YWHA-blocking peptide promoted germinal vesicle breakdown. This suggests that inhibiting the interactions between YWHA proteins and their binding partners releases the oocyte from meiotic arrest. Microinjection of isoform-specific, translation-blocking morpholino oligonucleotides to knockdown or downregulate YWHA protein synthesis in oocytes suggested a role for a specific YWHA isoform in maintaining the meiotic arrest. More definitively however, and in contrast to the knockdown experiments, oocyte-specific and global deletion of two isoforms of YWHA, YWHAH (14-3-3 eta) or YWHAE (14-3-3 epsilon) indicated that the complete absence of either or both isoforms does not alter oocyte development and release from the meiotic prophase I arrest.ConclusionsMultiple isoforms of the YWHA protein are expressed in mouse oocytes and eggs and interact with the cell cycle protein CDC25B, but YWHAH and YWHAE isoforms are not essential for normal mouse oocyte maturation, fertilization and early embryonic development.