Cdk1 Phosphorylation Research Articles

Identification and Screening of Selective WEE2 Inhibitors to Develop Non-Hormonal Contraceptives that Specifically Target Meiosis.

We used a progressive elimination strategy to identify oocyte-specific WEE2 kinase inhibitors for potential non-hormonal contraceptives that target meiosis. Beginning with an in-house library of over 300,000 compounds, virtual high throughput screening identified 57 WEE2 inhibitors with preferential predicted binding over the somatic variant WEE1. Seven compounds were further evaluated in vitro by enzyme-linked immunosorbent assay to measure biochemical inhibition on WEE1 and WEE2 phosphorylation of CDK1. To assess specificity, we evaluated WEE2-mediated inhibition of meiosis using in vitro oocyte fertilization, and WEE1-mediated inhibition of mitosis using a somatic cell proliferation assay. Our results from these assays identified three candidates for further development: 6-(2,6-dichlorophenyl)-2-((4-(2-(diethylamino)ethoxy) phenyl)amino)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (2), 6-(2,6-dichlorophenyl)-8-methyl-2-((4-morpholinophenyl) amino)pyrido[2,3-d]pyrimidin-7(8H)-one (12), and 3-((6-(2,6-dichlorophenyl)-8-methyl-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl)amino)benzoic acid (16).

Inactivated AMPK-α2 promotes the progression of diabetic brain damage by Cdk5 phosphorylation at Thr485 site

Changes in brain energy metabolism in diabetes mellitus, including increased insulin resistance and mitochondrial dysfunction, are critically involved in diabetes-related neurodegeneration, and associate with early cognitive impairment as well. The aim of this study is to detect the specific phosphorylated-Thr485- AMP-activated protein kinase (AMPK-α2), regulated by cyclin-dependent kinase 5 (Cdk5) paly the inhibitory functional role of AMPK-α2, Which is maybe the link to the accelerated diabetic brain damage progression. Here, we used GK rats, the type 2 diabetic animal model for in vivo studies and performed In vitro kinase assay, high glucose treatment, -phosphorylated mutation and protein expression in both HEK-293T and HT-22 cell lines. In vitro, the results show that murine wild-type AMPK-α2 was phosphorylated by Cdk5 at a (S/T)PX(K/H/R) phosphorylation consensus sequence, which was associated with decreased AMPK-α2 activity. Surprisingly, mutation of Thr485 to alanine in AMPK-α2 results in the abolished Cdk5 effects, demonstrating that Thr485-phosphorylation is critical to AMPK-α2 inhibition by Cdk5. In addition, these alterations in AMPK-α2-phosphorylation and -activity induced by Cdk5 is specific at Thr485. Furthermore, in GK rats, the increased phosphorylated- Thr 485 of AMPK-α2 results in the decreased AMPK-α2 activity, which is correlated with the apoptosis of neurons in hippocamps. After high glucose treatment, the decreased survival showed in AMPK-α2T485A HT-22 cells compared to AMPK-α2WT. The down-regulated of p-CREB, SNAP25, synaptophysin as well as synapsin-1were shown in both GK rats and HT-22 cell line. Meanwhile, pre-treated with either the specific Cdk5-inhibitor (roscovitine) or the antidiabetic AMPK-α2-inhibitor (metformin) could restore the alterations in neuronal protein expression. Our results suggest that Cdk5-mediated phosphorylated- Thr485 in AMPK-α2 may be involved in the pathogenesis of diabetic brain damage.

Aberrant activation of cyclin A-CDK induces G2/M-phase checkpoint in human cells

ABSTRACT Cyclin A-cyclin dependent kinase (CDK) activity is regulated by cyclin A proteolysis and CDK inhibitors (CKIs) during M and G1 phases. Our previous work has shown that constitutive activation of cyclin A-CDK in mouse somatic cells, by ectopic expression of stabilized human cyclin A2 (lacking the destruction box: CycAΔ80) in triple CKI (p21, p27, and p107)-knocked-out mouse embryonic fibroblasts, induces rapid tetraploidization. However, effects of such cyclin A-CDK hyperactivation in human cells have been unknown. Here, we show hyperactivity of cyclin A-CDK induces G2/M-phase arrest in human cell lines with relatively low expression of p21 and p27. Moreover, adenovirus E1A protein promoted CycAΔ80-derived G2/M-phase arrest by increasing the amount of cyclin A and cyclin A-CDK2 complex. This response was suppressed by an addition of ATR or Chk1 inhibitor. The amount of repressive phosphorylation of CDK1 at tyrosine 15 (Y15) was decreased by Chk1 inhibitor treatment. Moreover, we observed that co-expressing CDK1AF mutant, which is resistant to the repressive phosphorylation at threonine 14 and Y15, or cdc25A, which dephosphorylates CDK1 at Y15, suppressed the G2/M-phase arrest by CycAΔ80 with E1A. These results suggest that G2/M-phase arrest in human cells by hyperactivity of cyclin A-CDK2 is caused by repression of CDK1 via the cell cycle checkpoint ATR-Chk1 pathway.

CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle.

Telomere end-protection by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3’ single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow faithful telomere replication and to permit telomerase access to the 3’-end to solve the end replication problem. However, the regulation and physiological importance of t-loops in end-protection remains uncertain. Here, we identify a CDK phosphorylation site in the shelterin subunit, TRF2 (Ser365), whose dephosphorylation in S-phase by the PP6C/R3 phosphatase provides a narrow window during which the helicase RTEL1 is able to transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 on Ser365 outside of S-phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate ATM activation, but also counteracts replication conflicts at DNA secondary structures arising within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.

Leukemogenic Functions of Mutant ASXL1 Are Regulated By CDK-Mediated Phosphorylation

Additional sex combs-like 1 (ASXL1) is recurrently mutated in various myeloid neoplasms and its mutations confer poor prognosis. Most of the ASXL1 mutations in myeloid malignancies are frameshift or nonsense mutations, leading to expression of C-terminally truncated form of ASXL1 (ASXL1-MT). Recently, we have demonstrated that monoubiquitination of ASXL1-MT at lysine 351 plays an important role in enhancing the catalytic activity of BAP1, resulting in reduction of H2AK119ub and upregulation of posterior HOXA genes to promote myeloid leukemogenesis (Asada et al. Nat commun. 2018). However, roles of other post-translational modifications of ASXL1 including phosphorylation remain to be elucidated. In this study, we performed nano liquid chromatography tandem mass spectrometry (nano LC-MS/MS) and found that ASXL1 was phosphorylated at multiple serine (S) and threonine (T) residues including S503 residue, the major phosphorylated site of ASXL1. We generated an antibody that specifically recognizes phosphorylated S503 of ASXL1, and showed that CDK1/2 phosphorylated both wildtype ASXL1 and ASXL1-MT. However, phosphorylation status of ASXL1-MT at serine 503 did not affect its oncogenic activity. Thus, we considered the possibility that phosphorylations of other residues would compensate the effects of phosphorylation of ASXL1-MT at serine 503. Using public database and our data from mass spectrometry, we found additional five S/T residues containing a CDK phosphorylation motif (S/T-P). We then established a non-phosphomimetic mutant form of ASXL1-MT (ASXL1-MT-6A) whose six S/T residues were substituted to alanine. To compare the oncogenic functions between ASXL1-MT-6A and ASXL1-MT, we first assessed the impact of these mutants on differentiation of hematopoietic cells. Expression of ASXL1-MT-6A more strongly impaired differentiation of human CD34+ cord blood (CB) cells towards granulocytes and erythrocytes than that of ASXL1-MT. These effects of ASXL1-MT-6A on differentiation block were also observed in HL-60 cells. Next, we assessed the impact of phosphorylation of ASXL1-MT on its leukemogenic functions using RUNX1-ETO and N-Ras G12V-induced leukemia models, because RUNX1-ETO fusion or N-Ras mutations frequently coexist with ASXL1 mutations in patients with myeloid neoplasms. In agreement with our previous report, expression of ASXL1-MT enhanced proliferation of RUNX1-ETO expressing CB cells. ASXL1-MT-6A showed the stronger growth promoting effect than ASXL1-MT in RUNX1-ETO expressing CB cells in vitro by inhibiting apoptosis and progressing cell cycle. Moreover, ASXL1-MT-6A induced more rapid development of myeloid leukemia driven by N-Ras G12V than ASXL1-MT in vivo. ASXL1-MT-6A induced upregulation of Hoxa5, Hoxa7 and Hoxa9 more strongly than ASXL1-MT in myeloid progenitor cells. These data indicated that ASXL1-MT-6A is an active form of ASXL1-MT. Mechanistically, cycloheximide chasing assay revealed that ASXL1-MT-6A had increased protein stability than ASXL1-MT. Furthermore, cell cycle synchronization and immunoprecipitation assay demonstrated that degradation of ASXL1 protein began at early M phase by interacting with Cdc20, one of the coactivators of anaphase-promoting complex (APC/C), an E3 ubiquitin ligase complex involved in regulating cell cycle progression. Overexpression of Cdc20 promoted degradation of ASXL1, whereas depletion of Cdc20 using CRISPR-Cas9 system increased expression of ASXL1. These biochemical features were also observed for ASXL1-MT. Intriguingly, ASXL1-MT-6A showed resistance to degradation induced by APC/CCdc20. Therefore, CDK-mediated phosphorylation of ASXL1-MT triggers its degradation induced by APC/CCdc20. Collectively, we demonstrated that stability and leukemogenic functions of ASXL1-MT are regulated by CDK-mediated phosphorylation. Dynamics of protein degradation of ASXL1-MT triggered by phosphorylation and ubiquitination could be targets for therapeutic interventions of myeloid neoplasms harboring an ASXL1 mutation. Disclosures No relevant conflicts of interest to declare.

Phosphorylation of TFCP2L1 by CDK1 is required for stem cell pluripotency and bladder carcinogenesis

Molecular programs involved in embryogenesis are frequently upregulated in oncogenic dedifferentiation and metastasis. However, their precise roles and regulatory mechanisms remain elusive. Here, we showed that CDK1 phosphorylation of TFCP2L1, a pluripotency‐associated transcription factor, orchestrated pluripotency and cell‐cycling in embryonic stem cells (ESCs) and was aberrantly activated in aggressive bladder cancers (BCs). In murine ESCs, the protein interactome and transcription targets of Tfcp2l1 indicated its involvement in cell cycle regulation. Tfcp2l1 was phosphorylated at Thr177 by Cdk1, which affected ESC cell cycle progression, pluripotency, and differentiation. The CDK1‐TFCP2L1 pathway was activated in human BC cells, stimulating their proliferation, self‐renewal, and invasion. Lack of TFCP2L1 phosphorylation impaired the tumorigenic potency of BC cells in a xenograft model. In patients with BC, high co‐expression of TFCP2L1 and CDK1 was associated with unfavorable clinical characteristics including tumor grade, lymphovascular and muscularis propria invasion, and distant metastasis and was an independent prognostic factor for cancer‐specific survival. These findings demonstrate the molecular and clinical significance of CDK1‐mediated TFCP2L1 phosphorylation in stem cell pluripotency and in the tumorigenic stemness features associated with BC progression.

Glutamine synthetase facilitates cancer cells to recover from irradiation-induced G2/M arrest

ABSTRACTResistance to radiation of cancer cells can be either intrinsic or acquired, leading to treatment failure. In response to DNA damage caused by IR, cancer cells are arrested in cell cycle showing limited proliferation and increased apoptosis. However, radiation-resistant cells are able to overcome the cell cycle block and proceed to proliferation, for which the detailed mechanism remains to be elucidated. In the present study, we showed that radioresistant cells exhibited a recoverable G2/M phase during prolonged cell cycle and manifested lower apoptosis rate and more colony formation. RNA-seq analysis revealed that glutamine synthetase (GS, GLUL) gene was highly expressed in radioresistant cancer cells in comparison with the parental cells, which was in accordance with the G2/M arrest after ionizing radiation. Knocking out of GS in radioresistant cells resulted in a delayed G2/M recovery and lowered proliferation rate after ionizing radiation treatment, which was accompanied with increased inhibitory phosphorylation of CDK1 at Y15 and downregulated Cdc25B, a dual specific phosphatase of CDK1. Moreover, there was an enhanced complex formation of CDK1 and Cyclin B1 when the cells were rescued by re-introducing GS. In vivo, knocking down of GS significantly sensitized CNE2-R xenografts to RT in mice. In this study, we demonstrate a novel role of glutamine synthetase independent of metabolic function in promoting recovery from G2/M arrest caused by ionizing radiation, thus, causing cancer cell resistance to radiotherapy.

First meiotic anaphase requires Cep55-dependent inhibitory cyclin-dependent kinase 1 phosphorylation.

During mitosis, anaphase is triggered by anaphase-promoting complex (APC)-mediated destruction of securin and cyclin B1, which leads to inactivation of cyclin-dependent kinase 1 (Cdk1). By regulating APC activity, the mitotic spindle assembly checkpoint (SAC) therefore has robust control over anaphase timing to prevent chromosome mis-segregation. Mammalian oocytes are prone to aneuploidy, the reasons for which remain obscure. In mitosis, Cep55 is required post-anaphase for the final steps of cytokinesis. We found that Cep55-depleted mouse oocytes progress normally through early meiosis I, but that anaphase I fails as a result of persistent Cdk1 activity. Unexpectedly, Cdk1 inactivation was compromised following Cep55 depletion, despite on-time SAC silencing and intact APC-mediated proteolysis. We found that impaired Cdk1 inactivation was caused by inadequate inhibitory Cdk1 phosphorylation consequent upon failure to suppress Cdc25 phosphatase, identifying a proteolysis-independent step necessary for anaphase I. Thus, the SAC in oocytes does not exert exclusive control over anaphase I initiation, providing new insight into vulnerability to error.

The phosphatase PPM1A inhibits triple negative breast cancer growth by blocking cell cycle progression

Estrogen receptor (ER)-negative, progesterone receptor (PR)-negative and HER2-negative, or “triple negative,” breast cancer (TNBC) is a poor prognosis clinical subtype that occurs more frequently in younger women and is commonly treated with toxic chemotherapy. Effective targeted therapy for TNBC is urgently needed. Our previous studies have identified several kinases critical for TNBC growth. Since phosphatases regulate the function of kinase signaling pathways, we sought to identify critical growth-regulatory phosphatases that are expressed differentially in ER-negative, as compared to ER-positive, breast cancers. In this study, we examined the role of one of these differentially expressed phosphatases, the protein phosphatase Mg + 2/Mn + 2 dependent 1A (PPM1A) which is underexpressed in ER-negative breast cancer as compared to ER-positive breast cancers, in regulating TNBC growth. We found that PPM1A is deleted in ~40% of ER-negative breast cancers, and that induced expression of PPM1A suppresses in vitro and in vivo growth of TNBC cells. This study demonstrates that induction of PPM1A expression blocks the cell cycle and reduces CDK and Rb phosphorylation. These results suggest PPM1A is a crucial regulator of cell cycle progression in triple negative breast cancer. Our results also suggest that PPM1A loss should be explored as a predictive biomarker of CDK inhibitor sensitivity.

Tumor-specific inhibitory action of decorin on different hepatoma cell lines

BackgroundIn spite of therapeutic approaches, liver cancer is still one of the deadliest type of tumor in which tumor microenvironment may play an active role in the outcome of the disease. Decorin, a small leucine-rich proteoglycan is not only responsible for assembly and maintenance of the integrity of the extracellular matrix, but a natural inhibitor of cell surface receptors, thus it exerts antitumorigenic effects. Here we addressed the question whether this effect of decorin is independent of the tumor phenotypes including differentiation, proliferation and invasion. MethodFour hepatoma cell lines HepG2, Hep3B, HuH7 and HLE, possessing different molecular backgrounds, were selected to investigate. After proliferation tests, pRTK arrays, WB analyses, and immunofluorescent examinations were performed on decorin treated and control cells for comparison. ResultsSignificant growth inhibitory potential of decorin on three out of four hepatoma cell lines was proven, however the mode of its action was different. Induction of p21WAF1/CIP1, increased inactivation of c-myc and β-catenin, and decrease of EGFR, GSK3β and ERK1/2 phosphorylation levels were observed in HepG2 cells, pathways already well-described in literature. However, in the p53 deficient Hep3B and HuH7, InsR and IGF-1R were the main receptors transmitting signals. In harmony with its receptor status, Hep3B cells displayed high level of activated AKT. As the cell line is retinoblastoma mutant, ATR/Chk1/Wee1 system might hinder the cell cycle in G2/M phase via phosphorylation of CDK1. In Huh7 cells, all RTKs were inhibited by decorin followed by downregulation of AKT. Furthermore, HuH7 cell line responded with concentration-dependent ERK activation and increased phospho-c-myc level. Decorin had only a non-significant effect on the proliferation rate of HLE cell line. However, it responded with a significant decrease of pAKT, c-myc and β-catenin activity. In this special cell line, the inhibition of TGFβ may be the first step of the protective effect of decorin. ConclusionsBased on our results decorin may be a candidate therapeutic agent in the battle against liver cancer, but several questions need to be answered. It is certain that decorin is capable to exert its suppressor effect in hepatoma cells without respect to their phenotype and molecular background.

Abstract 3821: Taxane resistance in breast cancer controlled by the APC tumor suppressor

Abstract Adenomatous Polyposis Coli (APC) is a multi-domain tumor suppressor protein that binds to proteins including β-catenin, axin, and microtubules (MTs). APC is lost in many epithelial cancers and up to 70% of sporadic breast cancers, with a tendency towards triple negative breast cancers (TNBCs). In a mouse breast cancer model of APC loss, MMTV-PyMT;ApcMin/+, our laboratory previously demonstrated that APC loss resulted in metaplastic-like tumors, a subtype of TNBCs that often develops resistance to chemotherapy. Using the human breast cancer cell line, MDA-MB-157, we created APC knockdown cells (APCKD) using lentiviral mediated shRNA knockdown of APC, which demonstrated resistance to the Taxane family chemotherapeutic agent, Paclitaxel (PTX). Given that PTX and APC both alter MT dynamics, we sought to understand the molecular mechanisms of APC-mediated resistance. Based on the mechanism of action of PTX, we hypothesized that genes involved in the G2/M transition would be responsible for PTX resistance. We have also performed an unbiased analysis of transcriptomic changes downstream of APC loss to identify potential therapeutic targets to overcome PTX resistance. Cell cycle analysis demonstrated that the cell cycle profile of APCKD cells was not drastically changed after PTX treatment. However, PTX induction of p21 specifically in the APCKD cells suggests that APCKD cells may become senescent to avoid apoptosis. We next analyzed the expression of the G2/M checkpoint proteins, CDK1 and cyclin B1, by western blot. CDK1 expression was significantly increased in APCKD cells compared to control with no changes in CDK1 phosphorylation (Thr14, Thr161, Tyr15). Interestingly, there was no effect of PTX treatment in either cell line. Based on these findings, we performed combination studies using a CDK1 inhibitor (RO-3306) with PTX treatment. PARP (total and cleaved) was used to measure apoptosis, and showed enhanced cleavage in the APCKD cells specifically after combination treatment. In addition to the cell cycle analysis, we performed RNA-sequencing on parent and APCKD cells to understand the transcriptomic changes downstream of APC loss. Upon validation of results by qRT-PCR and western blot, we identified that APCKD cells had altered expression of ZNF366, ADAMTSL1, SELENBP1, and CYB5R2. These 4 genes are known to be involved in the development of breast cancer and response to chemotherapy. Ongoing studies in the laboratory will investigate the effect of manipulating expression of these genes. Overall, our studies strive to understand the molecular nature of PTX resistance in APCKD cells, and to identify a therapeutic target to work as a targeted treatment for some TNBC types. Citation Format: Emily Astarita, Megan Dieringer, Jenifer R. Prosperi. Taxane resistance in breast cancer controlled by the APC tumor suppressor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3821.

Abstract 4423: Antitumor activity of the novel oral highly selective Wee1 inhibitor Debio 0123

Abstract Background: The Wee1 tyrosine kinase is activated upon DNA damage and regulates the G2-M cell cycle checkpoint. Inhibition of Wee1, in conjunction with additional genetic alterations and/or addition of a DNA damaging agent, results in mitotic catastrophe and apoptosis of cancer cells, offering an attractive approach to treating cancer. The aim of the present study was to characterize the pharmacological properties of the newly discovered, orally available, and highly selective Wee1 inhibitor Debio 0123. Methods: Profiling of Debio 0123 was performed on 465 selected kinases in a cell-free system. Effects of Debio 0123 on downstream signaling were analyzed by ELISA and western blot in HT29 (colorectal adenocarcinoma) and A427 (lung carcinoma) cell lines. The in vitro growth inhibition activity of Debio 0123 was defined in a broad number of human cancer cell lines after 72h using a proliferation monolayer assay. Debio 0123 in vivo anti-tumoral activity was assessed in an A427 subcutaneous xenograft model in athymic nude mice. Debio 0123 pharmacodynamic effects were also evaluated after oral dosing in surrogate tissues of mice, rats and monkeys by immunohistochemistry. Results: Debio 0123 is a highly selective ATP-competitive Wee1 inhibitor with an IC50 in the low nanomolar range. Debio 0123 inhibited Cdk1 (Cdc2) phosphorylation at Y15, a direct substrate of Wee1 in cells. Debio 0123 also induced dose- and time-dependent increased mitosis and unrepaired DNA damage shown by increased phosphorylation of histone H3 and γH2AX foci formation. In vitro activity was further studied in a large panel of cancer cell lines. Debio 0123 demonstrated submicromolar cytotoxic activity on multiple cell lines from various histotypes. In vivo, Debio 0123 was shown to inhibit phosphorylation of Cdk1 in tissues of multiple species and induction of DNA damage in a xenograft model. When administered orally once daily for 28 consecutive days, Debio 0123 induced dose-dependent anti-tumor activity and was well tolerated at all doses tested. At 30 mg/kg, treatment with Debio 0123 resulted in tumor regression. Conclusion: Altogether these results demonstrate that Debio 0123 is a highly selective and potent Wee1 inhibitor able to prevent Cdk1 phosphorylation in multiple species and to induce apoptosis through accumulation of unrepaired DNA damage both in vitro and in vivo that result in tumor regression. The advancement of Debio 0123 into clinical studies may provide improved therapeutic outcomes for patients with cancer. Citation Format: Colin O'Dowd, Gerald Gavory, Frank Burkamp, Adam Treder, Caroline Boyd, Tim Harrison, Frederic Massière, Astrid Glück, Christophe Chardonnens, Andrea Zaffalon, Stefania Rigotti, Robert Mader, Grégoire Vuagniaux, Anne Vaslin Chessex. Antitumor activity of the novel oral highly selective Wee1 inhibitor Debio 0123 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4423.

CDK5 inhibits the clathrin-dependent internalization of TRPV1 by phosphorylating the clathrin adaptor protein AP2μ2.

Transient receptor potential vanilloid 1 (TRPV1), a nonselective, ligand-gated cation channel, responds to multiple noxious stimuli and is targeted by many kinases that influence its trafficking and activity. Studies on the internalization of TRPV1 have mainly focused on that induced by capsaicin or other agonists. Here, we report that constitutive internalization of TRPV1 occurred in a manner dependent on clathrin, dynamin, and adaptor protein complex 2 (AP2). The μ2 subunit of AP2 (AP2μ2) interacted directly with TRPV1 and was required for its constitutive internalization. Cyclin-dependent kinase 5 (CDK5) phosphorylated AP2μ2 at Ser45, which reduced the interaction between TRPV1 and AP2μ2, leading to decreased TRPV1 internalization. Intrathecal delivery of a cell-penetrating fusion peptide corresponding to the Cdk5 phosphorylation site in AP2μ2, which competed with AP2μ2 for phosphorylation by Cdk5, increased the abundance of TRPV1 on the surface of dorsal root ganglion neurons and reduced complete Freund's adjuvant (CFA)-induced inflammatory thermal hyperalgesia in rats. In addition to describing a mechanism of TRPV1 constitutive internalization and its inhibition by CDK5, these findings demonstrate that CDK5 promotes inflammatory thermal hyperalgesia by reducing TRPV1 internalization, providing previously unidentified insights into the search for drug targets to treat pain.

Combined Aurora Kinase A (AURKA) and WEE1 Inhibition Demonstrates Synergistic Antitumor Effect in Squamous Cell Carcinoma of the Head and Neck.

Human papillomavirus (HPV)-negative head and neck squamous cell carcinomas (HNSCC) commonly bear disruptive mutations in TP53, resulting in treatment resistance. In these patients, direct targeting of p53 has not been successful, but synthetic lethal approaches have promise. Although Aurora A kinase (AURKA) is overexpressed and an oncogenic driver, its inhibition has only modest clinical effects in HPV-negative HNSCC. We explored a novel combination of AURKA and WEE1 inhibition to overcome intrinsic resistance to AURKA inhibition.Experimental Design: AURKA protein expression was determined by fluorescence-based automated quantitative analysis of patient specimens and correlated with survival. We evaluated treatment with the AURKA inhibitor alisertib (MLN8237) and the WEE1 inhibitor adavosertib (AZD1775), alone or in combination, using in vitro and in vivo HNSCC models. Elevated nuclear AURKA correlated with worse survival among patients with p16(-) HNSCC. Alisertib caused spindle defects, G2-M arrest and inhibitory CDK1 phosphorylation, and cytostasis in TP53 mutant HNSCC FaDu and UNC7 cells. Addition of adavosertib to alisertib instead triggered mitotic entry and mitotic catastrophe. Moreover, in FaDu and Detroit 562 xenografts, this combination demonstrated synergistic effects on tumor growth and extended overall survival compared with either vehicle or single-agent treatment. Combinatorial treatment with adavosertib and alisertib leads to synergistic antitumor effects in in vitro and in vivo HNSCC models. These findings suggest a novel rational combination, providing a promising therapeutic avenue for TP53-mutated cancers.

Inhibition of cyclin-dependent kinases by AT7519 is effective to overcome chemoresistance in colon and cervical cancer

Cyclin-dependent kinases (CDK), a family of heterodimeric kinases that play central roles in regulation of cell cycle progression and transcription, have garnered attention in recent years because their aberrant activity has been reported in a wide variety of human cancers. AT7519 is a multitargeted CDK inhibitor that is currently in clinical trials for the treatment of refractory blood cancers. In this work, we are the first to provide preclinical evidence that AT7519 is an attractive candidate to overcome chemoresistance in colon and cervical cancer. We show that AT7519 is effective in targeting a panel of colon and cervical cancer cell lines, with IC50 range from 0.1 to 1 μM. Importantly, AT7519 at similar IC50 range inhibits growth and induces apoptosis of paclitaxel-resistant cervical cancer cells and 5-FU-resistant colon cancer cells. AT7519 at sublethal concentration remarkably augments the inhibitory effects of 5-FU and paclitaxel in colon and cervical cancer cells. Mechanistically, we show that AT7519 suppresses phosphorylation of CDK1, CDK2 and RNA polymerase II in chemoresistant colon and cervical cancer cells. We further confirm the efficacy of AT7519 and its mechanisms of the action using two independent chemoresistant xenograft mouse models: 5-FU-resistant colony cancer xenograft and paclitaxel-resistant cervical cancer xenograft. Our findings support the clinical trials of AT7519 for cancer treatment. Our work also demonstrates the therapeutic value of inhibiting CDK in chemoresistant cancers.

Characterization of Cks2 localization and interaction with Cdk1 in mitotic cells of Xenopus laevis embryos

Although mitotic regulation has been extensively studied, several of the mechanisms behind this process have yet to be fully understood. Our group is particularly interested in mechanisms regulating the timing of mitotic exit and how such mechanisms coordinate with other mitotic cues such as spindle orientation. Recent work in our lab has shown that spindle movements in epithelial cells of the developing frog embryo correlate with mitotic exit and perturbations in normal spindle movements lengthen metaphase. Further, these changes in mitotic progression are accompanied by increases in Cdk1 Tyr15 phosphorylation, specifically at cell junctions. We propose that the link between spindle movement and mitotic progression is an important part of the mechanisms influencing spindle orientation in the frog epithelium. Our current work examines the role of Cks2 in the aforementioned mechanism. Cks2 serves as a targeting subunit for the mitotic kinase Cdk1. Specifically, work in yeast suggests Cks2 may direct Cdk1 toward the anaphase‐promoting complex (APC) and interact with both complexes, facilitating the phosphorylation of Cdk1 and activation of the APC and thus help control mitotic exit. In this study, we characterize Cks2's mitotic function in Xenopus laevis. Our aim is to characterize Cks2 distribution throughout mitosis and determine whether perturbing Cks2 function alters mitotic progression in the gastrula‐stage epithelium. We hypothesize that if Cks2 influences Cdk1 function in mitosis, then the two proteins will exhibit similar localization patterns in mitotic cells. We visualized endogenous Cks2 and also expressed GFP‐tagged Cks2 in fixed epithelial tissues of Xenopus laevis embryos through confocal microscopy. Our early data show that Cks2 localization overlaps with that of a phosphorylated Cdk1(pY15) at cell junctions and overexpression of GFP‐tagged Cks2 results in increased levels of Cdk1‐pY15. In addition, the assay expressing a dominant negative version of Cks2 suggests that depletion of endogenous Cks2 activity may contribute to decreased Cdk1‐pY15 at cell junctions and increased cell area. Therefore, these data suggest a link between Cks2 and Cdk1 in mitotic regulation and are a first step toward a greater characterization of Cks2's role in regulating mitotic progression in vertebrate organisms.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Cdk5 in Kalirin7-Mediated Formation of Dendritic Spines.

A majority of excitatory synapses in the brain are localized on the dendritic spines. Alterations of spine density and morphology are associated with many neurological diseases. Understanding the molecular mechanisms underlying spine formation is important for understanding these diseases. Kalirin7 (Kal-7) is localized to the postsynaptic side of excitatory synapses in the neurons. Overexpression of Kal-7 causes an increase in spine density whereas knockdown expression of endogenous Kal-7 results in a decrease in spine density in primary cultured cortical neurons. However, the mechanisms underlying Kal-7-mediated spine formation are not entirely clear. Cyclin-dependent kinase 5 (Cdk5) plays a vital role in the formation of spines and synaptic plasticity. Kal-7 is phosphorylated by CDK5 at Thr1590, the unique Cdk5 phosphorylation site in the Kal-7 protein. This study was to explore the role of CDK5-mediated phosphorylation of Kal-7 in spine formation and the underlying mechanisms. Our results showed expression of Kal-7T/D (mimicked phosphorylation), Kal-7T/A mutants (blocked phosphorylation) or wild-type (Wt) Kal-7 caused in a similar increase in spine density, while spine size of Wt Kal-7-expressing cortical neurons was bigger than that in Kal-7 T\A-expressing neurons, but smaller than that in Kal-7T/D-expressing neurons. The fluorescence intensity of NMDA receptor subunit NR2B (GluN2B) staining was stronger along the MAP2 positive dendrites of Kal-7T/D-expressing neurons than that in Kal-7T/A- or Wt Kal-7-expressing neurons. The fluorescence intensity of AMPA receptor subunit GluR1 (GluA1) staining showed the same trend as GluN2B staining. These findings suggest that Cdk5 affects the function of Kal-7 on spine morphology and function via GluN2B and GluA1 receptors during dendritic spine formation.

CDK7 inhibitor suppresses tumor progression through blocking the cell cycle at the G2/M phase and inhibiting transcriptional activity in cervical cancer.

BackgroundThe disordered cell cycle and dysregulated expression of numerous oncogenes involved in tumor-relevant processes are highly related to the tumorigenesis of cervical cancer. Cyclin-dependent kinase 7 (CDK7) constitutes the indispensable catalytic subunit of CDK-activating kinase (CAK), which is required for both cell cycle transition and transcriptional regulation. However, research regarding the antitumor effects of CDK7 inhibition in cervical cancer remains unclear.PurposeOur study aims to explore the antineoplastic effects of the CDK7 inhibitor THZ1 in cervical cancer cells and to find a potential agent for cervical cancer treatment.MethodsThe CRISPR-Cas9 system was used to knock down CDK7. The Cell Counting Kit-8 (CCK-8) assay was used to detect the cell viability after CDK7 depletion and THZ1 treatment. Western blot was employed to detect protein expression. The expression levels of mRNA were assayed through qRT-PCR. Flow cytometry analysis was used to assay the apoptotic cells and cell cycle distribution. Gene expression microarray analysis was used to identify the differential expression of the genes. Subcutaneous xenograft mouse model was performed to test the antineoplastic effects of THZ1 in vivo.ResultsWe revealed that the genetic depletion of CDK7 using the CRISPR-Cas9 system exhibited great cell growth inhibition in cervical cancer cell lines, consistent with the effects of CDK7 blocking using THZ1. Cervical cancer cells were highly sensitive to THZ1 treatment, and a low concentration of THZ1 could induce substantial cell apoptosis. THZ1 specifically perturbed the phosphorylation of cell cycle regulator CDK1 and decreased the expression of cyclin B1, leading to a cell cycle blockage at the G2/M phase and inducing cell growth inhibition. The gene expression microarray analysis showed that massive oncogene transcripts, especially those associated with tumorigenesis, were preferential suppressed after THZ1 treatment. The qRT-PCR confirmed that several essential oncogenes in tumorigenesis (c-MYC, hTERT, RAD51, and BCL-2) and HPV viral oncogenes (E6 and E7) were preferentially repressed by THZ1. Moreover, THZ1 exhibited substantial antineoplastic effects against cervical cancer in vivo without inducing obvious side effects.ConclusionThese findings indicated that the CDK7 inhibitor THZ1 is a potential option in cervical cancer treatment owing to its ability to inhibit cell cycle progression and transcriptional activity.

Abstract PD7-12: Inhibition of CDK7 overcomes resistance to CDK4/6 inhibitors in hormone receptor positive breast cancer cells

Abstract Background: Despite the efficacy of the combinations of endocrine therapy (ET) and CDK4/6 inhibitors in the hormone receptor positive (HR+) metastatic breast cancer (BC) setting, the majority of patients eventually acquire resistance to these treatments. The loss of Rb activity is an important mechanism of acquired resistance to CDK4/6 inhibitors. Preclinical and clinical data support the RB1 genetic aberrations, including mutations and deletions with loss of expression, as mechanism of palbociclib (palbo) resistance. To study vulnerabilities and potential treatment targets to overcome resistance to CDK4/6 inhibitors associated with Rb loss we studied a T47D palbo-resistant (T47D PDR) HR+ BC cell model obtained by exposing palbo-sensitive cells to increasing concentrations of palbo. These cells harbour deletion of RB1, and show increased cyclin E1 and decreased ER expression. Methods: To identify genes that are essential for cell growth and genes whose loss enhances cell growth in palbo-resistant cells with loss of Rb, we performed genome-wide CRISPR-Cas9 knockout (KO) screens in T47D palbo-sensitive (T47D PDS) and T47D PDR cells. We searched for significantly essential genes that are targets with available drugs and tested the efficacy of these compounds in T47D PDS and T47D PDR cells. Results: By employing a genome wide CRISPR KO screen, we identified 29 genes that were significantly positively selected in the T47D PDR cells. Among these genes were known tumor suppressor genes, such as TSC2 (β score: 0.89) and PTEN (β score: 0.68). We identified close to 600 genes that were significantly essential (negatively selected) for T47D PDR growth. CDK4, CDK6 and CCND1 were essential in the T47D PDS parental cells but lost essentiality with the acquisition of palbo resistance (FDR> 0.05). CDK7 (β score: -1.21) and CDK2 (β score: -1.67) were among the top ranked essential genes (FDR: 0). CDK7 is a transcriptional CDK and has CDK activating kinase (CAK) activity. The CAK activity includes phosphorylation of CDK2 and CDK9. To follow up on our CRISPR screen results we tested the activity of the selective CDK7 inhibitors SY-1365 (currently in phase 1 clinical development) and THZ1 in the T47D PDS and T47D PDR cells. We saw dose dependent activity in both cell line models. Moreover, the IC50 values were comparable in T47D PDS and T47D PDR cells (THZ1; PDS: 9.93 nM, PDR with Palbo: 32.8 nM, PDR without Palbo: 1.08 nM) (SY1365; PDS: 1.60 nM, PDR with Palbo: 6.70 nM, PDR without Palbo: 1.87 nM). Combination studies of SY-1365 and fulvestrant in T47D PDR showed synergistic activity at lower concentrations. Conclusions: In a model of palbo resistance with loss of Rb we found that cyclin D1, CDK4 and CDK6 are not essential, suggesting cross-resistance to other CDK4/6 inhibitors in this setting. We identified CDK7 as a significant essential gene and potential therapeutic target. We validated these findings with the selective CDK7 inhibitors, THZ1 and SY-1365. Our results offer a new therapeutic strategy for the treatment of palbo-resistant HR+ BC. Citation Format: Guarducci C, Nardone A, Feiglin A, Migliaccio I, Malorni L, Bonechi M, Benelli M, Di Leo A, Hodgson G, Shapiro G, Brown M, Jeselsohn R. Inhibition of CDK7 overcomes resistance to CDK4/6 inhibitors in hormone receptor positive breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD7-12.

A Code of Mono-phosphorylation Modulates the Function of RB

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.