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Triple negative breast cancer (TNBC) is the most aggressive subtype of all breast cancers. However, unlike other breast cancer subtypes, current treatments for TNBC are restricted and this scarcity of viable options is the key contributor to the poorer prognosis. Despite early response, almost all the targeted drugs tested in TNBC eventually fail due to the development of resistance. Patients’ data have shown that the expression level of Mediator kinases CDK8 and CDK19 are elevated in TNBC, and that higher expression of CDK8/19 is correlated with more advanced diseases and worse prognosis. Selective CDK8/19 inhibitor SNX631, when used as a single agent, inhibited the growth of several TNBC cell lines in vitro, cell-derived xenografts (CDXs) as well as patient derived xenografts (PDXs) in vivo, suggesting the potential of targeting CDK8/19 in treating TNBC. We also analyzed the effect of CDK8/19 inhibition on the outcome of treatment with mTORC1 inhibitor everolimus (RAD001), an approved drug for several cancers with mutations of PTEN or PI3KCA. SNX631 exhibited a synergistic effect in combination with everolimus on suppressing TNBC cell growth in vitro. In vivo treatment with everolimus alone achieved a strong tumor growth inhibition in TNBC xenograft models but all the tumors eventually resumed growth, indicated the development of resistance. Significantly, the addition of a CDK8/19 inhibitor prevented the emergence of in vivo everolimus resistance both in CDX and PDX tumors upon treatment for up to 150 days, suggesting a potential for extending remission or even achieving cures in TNBC. Transcriptomic analysis demonstrated that this effect was due to the prevention of transcriptional reprogramming associated with everolimus resistance in tumor cells. Citation Format: Xiaokai Ding, Hao Ji, Amanda C. Sharko, Juergen Loskutov, Zachary Mack, Jadyn Myers, Mengqian Chen, Elena Pugacheva, Igor Roninson, Eugenia Broude. PD5-10 Dual therapeutic targeting of CDK8/19 and mTOR in triple negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-10.

The Senexins are potent and selective quinazoline inhibitors of CDK8/19 Mediator kinases. To improve their potency and metabolic stability, quinoline-based derivatives were designed through a structure-guided strategy based on the simulated drug-target docking model of Senexin A and B. A library of quinoline-Senexin derivatives was synthesized to explore the structure-activity relationship. An optimized compound 20a (Senexin C) exhibits potent CDK8/19 inhibitory activity with a high selectivity. Senexin C is more metabolically stable and provides a more sustained inhibition of CDK8/19-dependent cellular gene expression when compared with the prototype inhibitor Senexin B. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) evaluation using a novel tumor-based PD assay showed good oral bioavailability of Senexin C with a strong tumor-enrichment PK profile and tumor-PD marker responses. Senexin C inhibits MV4-11 leukemia growth in a systemic in vivo model with good tolerability.

Supplemental data for SMAD4, CTNNB1 and STAT1 knockdown and microRNA analysis

List of key resources and reagents

Unresectable hepatic metastases of colon cancer respond poorly to existing therapies and are a major cause of colon cancer lethality. In this study, we evaluated the therapeutic viability of targeting the mediator kinase CDK8, an early clinical stage drug target, as a means to suppress metastasis of colon cancer. CDK8 was amplified or overexpressed in many colon cancers and CDK8 expression correlated with shorter patient survival. Knockdown or inhibition of CDK8 had little effect on colon cancer cell growth but suppressed metastatic growth of mouse and human colon cancer cells in the liver. This effect was due in part to inhibition of already established hepatic metastases, indicating therapeutic potential of CDK8 inhibitors in the metastatic setting. In contrast, knockdown or inhibition of CDK8 had no significant effect on the growth of tumors implanted subcutaneously, intrasplenically, or orthotopically in the cecum. CDK8 mediated colon cancer growth in the liver through downregulation of matrix metalloproteinase (MMP) inhibitor TIMP3 via TGFβ/SMAD-driven expression of a TIMP3-targeting microRNA, miR-181b, along with induction of Mmp3 in murine or MMP9 in human colon cancer cells via Wnt/β-catenin-driven transcription. These findings reveal a new mechanism for negative regulation of gene expression by CDK8 and a site-specific role for CDK8 in colon cancer hepatic metastasis. Our results indicate the utility of CDK8 inhibitors for the treatment of colon cancer metastases in the liver and suggest that CDK8 inhibitors may be considered in other therapeutic settings involving TGFβ/SMAD or Wnt/β-catenin pathway activation.Significance:These findings demonstrate that inhibition of the transcription-regulating kinase CDK8 exerts a site-specific tumor-suppressive effect on colon cancer growth in the liver, representing a unique therapeutic opportunity for the treatment of advanced colon cancer.

The objective of this study was to investigate the effects of iron nanoliposomes on iron supplementation and toxicity in SD rats induced by a low-iron diet. The size and infrared spectroscopy of a liposomal oral delivery system were investigated. The particle size of nanoliposomes embedded with chelates was increased. Infrared spectra proved that peptides-iron and blank nanoliposomes were bonded by interaction forces, including the fracture of hydrogen bonds, C = C bonds, hydrophobic interaction, and C-N bonds. We found that iron supplementation chelates had a certain protective effect on viscera after being embedded by nanoliposomes. After 10 days of treatment, the concentration of hemoglobin could be gradually increased. Nanoliposome encapsulated peptides-iron has a better effect than other groups. At the same time, SOD, MDA, and CAT reached normal levels after 20 days. Histological results showed that the sections of the nanoliposomes groups were clearer than those of the other groups. There was a little inflammation in the liver without obvious pathological changes, which also proved that the iron chelates embedded by nanoliposomes had no obvious side effects on iron supplementation in rats. Nanoliposome encapsulated peptides-iron has a small side effect and a significant curative effect of iron supplementation. It maybe has a good application prospect in the clinical medical field.

Breast cancers (BrCas) that overexpress oncogenic tyrosine kinase receptor HER2 are treated with HER2-targeting antibodies (such as trastuzumab) or small-molecule kinase inhibitors (such as lapatinib). However, most patients with metastatic HER2+BrCa have intrinsic resistance and nearly all eventually become resistant to HER2-targeting therapy. Resistance to HER2-targeting drugs frequently involves transcriptional reprogramming associated with constitutive activation of different signaling pathways. We have investigated the role of CDK8/19 Mediator kinase, a regulator of transcriptional reprogramming, in the response of HER2+BrCa to HER2-targeting drugs. CDK8 was in the top 1% of all genes ranked by correlation with shorter relapse-free survival among treated HER2+BrCa patients. Selective CDK8/19 inhibitors (senexin B and SNX631) showed synergistic interactions with lapatinib and trastuzumab in a panel of HER2+BrCa cell lines, overcoming and preventing resistance to HER2-targeting drugs. The synergistic effects were mediated in part through the PI3K/AKT/mTOR pathway and reduced by PI3K inhibition. Combination of HER2- and CDK8/19-targeting agents inhibited STAT1 and STAT3 phosphorylation at S727 and up-regulated tumor suppressor BTG2. The growth of xenograft tumors formed by lapatinib-sensitive or -resistant HER2+breast cancer cells was partially inhibited by SNX631 alone and strongly suppressed by the combination of SNX631 and lapatinib, overcoming lapatinib resistance. These effects were associated with decreased tumor cell proliferation and altered recruitment of stromal components to the xenograft tumors. These results suggest potential clinical benefit of combining HER2- and CDK8/19-targeting drugs in the treatment of metastatic HER2+BrCa.

Metabolic reprogramming is a hallmark in multiple types of malignancies. Fast-growing cancer cells require facilitated synthesis of essential metabolites and excessive energy production. However, whether they are internally coordinated remains largely unknown. Herein, we found that de novo pyrimidine synthesis enhanced aerobic glycolysis in cancer cells. Mechanistically, pyrimidine biosynthesis augmented Notch signaling and transcriptionally increased c-Myc expression, leading to up-regulation of critical glycolytic enzymes. Further studies revealed that pyrimidine synthesis could stabilize γ-secretase subunit Nicastrin at post-translational N-linked glycosylation level, thereby inducing the cleavage and activation of Notch. Besides, we found that up-regulation of the key enzymes for de novo pyrimidine synthesis CAD and DHODH conferred the chemotherapeutic resistance of gastric cancer via accelerating glycolysis, and pharmacologic inhibition of pyrimidine biosynthetic pathway sensitized cancer cells to chemotherapy in vitro and in vivo. Collectively, our findings provide more insights into the regulation of aerobic glycolysis and a metabolic vulnerability that can be exploited to enhance chemotherapy efficacy in gastric cancer.

Yes-associated protein (YAP) is a vital transcriptional co-activator that activates cell proliferation and evasion of apoptosis for the promotion of tumorigenesis. The von Hippel-Lindau tumor suppressor protein (pVHL), as a critical component of E3 ubiquitin ligase, targets various substrates to regulate tumor progression. However, the precise molecular mechanisms of pVHL during tumorigenesis remain largely unclear. Herein, we found that there was a significant negative correlation between pVHL and YAP at protein level in the TCGA-LUAD dataset and our cohort. Over-expression of pVHL decreased YAP protein expression and reduced its transcriptional activity. Further study indicated that pVHL did not affect YAP mRNA level but decreased YAP protein stability in a lysosome-dependent manner. In addition, the pVHL-mediated degradation of YAP inhibited cellular proliferation, migration, and enhanced chemosensitivity to cisplatin in lung adenocarcinoma cells. Interestingly, the pVHL-mediated YAP degradation was blocked by elevated O-GlcNAcylation. Collectively, our findings demonstrate that pVHL modulates the lysosomal degradation of YAP, and may provide more clues to better understanding the tumor suppressive effects of pVHL.

Liver fibrosis is a continuous wound healing response caused by chronic liver injury, and the activation of hepatic stellate cells (HSCs) is considered as the main event for it. Core fucosylation catalyzed by FUT8 refers to adding the fucosyl moiety to the innermost GlcNAc residue of N-linked oligosaccharides and is involved in many biological processes such as cell differentiation, migration, and signaling transduction. Aberrant core fucosylation is associated with a variety of diseases including cardiovascular disease, tumors and neuroinflammation, but much less is understood in liver fibrosis. Herein, we reported FUT8 mRNA level was increased in patients with liver fibrosis from GEO database and positively correlated with fibrosis progression. FUT8 expression and the core fucosylation were also elevated in TAA-induced mouse liver fibrosis model, and were mainly distributed in the fibrous septum of mouse liver. TGF-β1, as the most pro-fibrogenic cytokine, could promote the expression of FUT8 and total core fucosylation levels in HSCs in vitro. However, up-regulation of FUT8 in turn inhibited TGF-β1-induced trans-differentiation, migration and pro-fibrogenic signaling pathways in HSCs. In conclusion, our results suggest that the up-regulation of FUT8 inhibits TGF-β1-induced HSC activation in a negative feedback loop, and provide potential new therapeutic strategy for liver fibrosis by targeting FUT8.

Following androgen deprivation therapy (ADT), prostate cancers (PCa) progress to castration resistant prostate cancer (CRPC) status, independent of androgen signaling, through changes in androgen receptor (AR) or by converting to AR-negative forms. Metastatic CRPC remains incurable, and the most potent new drugs prolong survival by only a few months. There is an urgent need for novel agents against advanced CRPC. CDK8 and CDK19 are two isoforms of Mediator kinase, which regulates transcriptional reprogramming, a key process in cancer development, metastasis, and drug resistance. Bioinformatic analysis of clinical prostate cancers shows that CDK19 expression increases during PCa development and progression reaching higher levels than in any other cancers, whereas CDK8 expression increases when PCa becomes CRPC. We have investigated the effects of CDK8/19 Mediator kinase inhibition on gene expression and tumor growth in several CPRC tumor models, growing subcutaneously or in the bone, the principal cause of PCa lethality. CDK8/19 inhibition decreased androgen-stimulated expression of the most strongly androgen-regulated genes, including PSA, in androgen-responsive PCa in vitro and in vivo. CRISPR knockout of both CDK8 and CDK19 in 22Rv1 CRPC cells that express both full-length AR and its androgen-independent V7 variant, had little effect on the tumor growth in intact male mice but strongly suppressed tumor growth in castrated animals. Moreover, re-expression of CDK19 but not of its kinase-dead mutant partially restored tumor growth in castrated mice. Similarly, SNX631, a selective CDK8/19 inhibitor, had little effect on 22Rv1 growth in intact mice but strongly suppressed the growth of these cells (but not of their knockout derivative) in castrated mice. Prolonged (up to 300 days) treatment with SNX631 induced tumor shrinkage and disappearance in a subset of 22Rv1 tumors grown in castrated animals. Transcriptomic analysis revealed that CDK8/19 inhibition or knockout suppressed castration-induced transcriptional reprogramming in tumor cells and affected stromal gene expression. SNX631 also inhibited the growth of a CRPC PDX derived from a PCa patient who failed casodex, abiraterone, and docetaxel, in castrated mice. SNX631 also significantly inhibited in vivo growth of AR-negative PC3 cells both in the flank and in the bone, the principal metastatic site of PCa. These results warrant the development of CDK8/19 inhibitors for the presently incurable metastatic CRPC. Funding acknowledgement: This research was funded by NIH grant R44CA203184 (M.C., I.R., M.L., Y.S.) Citation Format: Jing Li, Thomas Hilimire, Chen Cheng, Eugenia V. Broude, Yueying Liu, Michael B. Lilly, Yusuke Shiozawa, George Wilding, Igor B. Roninson, Mengqian Chen. Inhibition of CDK8/19 mediator kinase suppresses primary and metastatic growth of castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2357.

Energy-efficient technologies such as earth-to-air heat exchangers (EAHEs) and buoyancy-driven natural ventilation (BV) are employed for space conditioning. However, a fan is required in conventional EAHEs for air circulation, and BV normally serves as a passive cooling measure in temperate transitional seasons. This paper proposes the coupling of EAHEs and the buoyancy generated inside a building to achieve passive and autonomous ventilation without requiring any mechanical system. A model is developed to investigate the effects of the coupled system, with a primary focus on the dynamics of the airflow temperature, flow rate, and cooling/heating capacity provision. The model results are in good agreement with those of the computational fluid dynamics simulation. The model is applied in a hypothetical building located in a hot-summer/cold-winter region (Chongqing, China). The proposed coupled scheme is superior to the BV in hot and cold seasons. The indoor air temperature, ventilation flow rate, and cooling/heating capacity are found to fluctuate asynchronously. The cooling capacity is 56.3 kWh for the hottest day, and the heating capacity is 111.1 kWh for the coldest day. The maximum cooling or heating capacity is nearly achieved at the hottest or coldest times with the help of ventilation flow rate fluctuation.

A gene (aga0917) encoding a putative β-agarase was identified from the genome of Pseudoalteromonas fuliginea YTW1-15-1. The nucleotide sequence analysis revealed that aga0917 had significant homology to the agarase genes of the GH16 family. aga0917 encodes a putative protein of 290 amino acids with an estimated molecular mass of 32.5 kDa, including a 21-amino acid signal peptide. A gene fragment encoding only the putative mature form of Aga0917 (269 amino acids) was overexpressed in Escherichia coli BL21 (DE3) pLysS as a 6 × histine-tagged fusion protein (rmAga0917). The Km, Vmax, and kcat for agarose of rmAga0917 were 39.6 mg/mL, 334 (U/mg) of protein, and 178 (1/s), respectively. According to the results of thin-layer chromatography and mass spectrometry analysis, the main end product from agarose with rmAga0917 was neoagarotetraose, in addition to a small amount of neoagarobiose. Notably, the recombinant protein rmAga0917 showed optimum activity at 60°C and retained approximately 100% agarolytic activity after being kept at 40°C for 1 h and 57% residual activity after incubation at 50°C for 1 h. The rmAga0917 exhibited maximum agarase activity at pH 6.0, and retained more than 80% of activity after incubation over a range of pH 4.0-9.0 for 1 h at 4°C.

The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB include IL8, CXCL1, and CXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.

We introduce a multivariate heteroscedastic measurement error model for replications under scale mixtures of normal distribution. The model can provide a robust analysis and can be viewed as a generalization of multiple linear regression from both model structure and distribution assumption. An efficient method based on Markov Chain Monte Carlo is developed for parameter estimation. The deviance information criterion and the conditional predictive ordinates are used as model selection criteria. Simulation studies show robust inference behaviours of the model against both misspecification of distributions and outliers. We work out an illustrative example with a real data set on measurements of plant root decomposition.

CDK8 and CDK19 Mediator kinases are transcriptional co-regulators implicated in several types of cancer. Small-molecule CDK8/19 inhibitors have recently entered or are entering clinical trials, starting with breast cancer and acute myeloid leukemia (AML). To identify other cancers where these novel drugs may provide benefit, we queried genomic and transcriptomic databases for potential impact of CDK8, CDK19, or their binding partner CCNC. sgRNA analysis of a panel of tumor cell lines showed that most tumor types represented in the panel, except for some central nervous system tumors, were not dependent on these genes. In contrast, analysis of clinical samples for alterations in these genes revealed a high frequency of gene amplification in two highly aggressive subtypes of prostate cancer and in some cancers of the GI tract, breast, bladder, and sarcomas. Analysis of survival correlations identified a group of cancers where CDK8 expression correlated with shorter survival (notably breast, prostate, cervical cancers, and esophageal adenocarcinoma). In some cancers (AML, melanoma, ovarian, and others), such correlations were limited to samples with a below-median tumor mutation burden. These results suggest that Mediator kinases are especially important in cancers that are driven primarily by transcriptional rather than mutational changes and warrant an investigation of their role in additional cancer types.

One of every five breast cancers are driven by gene amplification and overexpression of HER2/ERBB2 tyrosine kinase. HER2+ breast cancers are treated both by monoclonal antibodies (such as trastuzumab) and small-molecule HER2 kinase inhibitors (such as lapatinib). However, some HER2+ tumors are inherently resistant to HER2-targeting drugs and most of metastatic cancers treated with such drugs eventually acquire resistance. We have analyzed the effect of small-molecule inhibitors of transcriptional serine/threonine kinases CDK8 and CDK19, paralogs of the Mediator kinase that modulates the activity of several signal-responsive transcription factors, on the response to lapatinib in a panel of HER2+ breast cancer cell lines. Selective CDK8/19 inhibitors senexin B and SNX631 showed in vitro synergy with lapatinib in different lapatinib-responsive cell lines and overcame lapatinib resistance in cells with either inherent or acquired resistance to this drug. In vivo treatment with SNX631 significantly inhibited the growth of cell-line based and a patient-derived xenograft (PDX) models of HER2+ breast cancer. A combination of SNX631 and lapatinib drastically suppressed the growth of tumors formed by lapatinib-sensitive HCC1954 cells and their lapatinib-resistant derivative HCC1954-L, overcoming lapatinib resistance of the latter. GSEA analysis of RNA-Seq data from cell treated in vitro with lapatinib, senexin B or their combination, showed that lapatinib activated the JAK/STAT pathway but the addition of the CDK8/19 inhibitor suppressed such induction. CDK8/19 inhibitors, when combined with lapatinib, suppressed STAT3 and STAT1 phosphorylation at Ser727. CRISPR/CAS9 knockout of STAT1 and STAT3 together (but not individually) sensitized HER2+ breast cancer cells to lapatinib and reduced the lapatinib-sensitizing effect of CDK8/19 inhibitors, suggesting that STAT1 and STAT3 mediate the effect of CDK8/19. Through miRNA-Seq and QPCR analysis, we have also identified two miRNAs that are regulated by STAT1 or STAT3 and overexpressed in lapatinib-resistant cells. These miRNAs are upregulated upon lapatinib treatment, whereas the combination of lapatinib with senexin B decreases their expression, suggesting that these miRNAs may mediate the effects of CDK8/19 and STAT1/3 on lapatinib resistance. Our results suggest that combining HER2- and CDK8/19-targeting drugs could be a promising therapeutic direction for HER2+ breast cancers after the failure of HER2-targeted therapy. Citation Format: Xiaokai Ding, Amanda C. Sharko, Martina S-J McDermott, Hao Ji, Alexander Chumanevich, Gary P. Schools, Zachary T. Mack, Elena Pugacheva, Victor Tatarskiy, Anastasia Khrustaleva, Alexander Tyakht, Michael Shtutman, Mengqian Chen, Igor Roninson, Eugenia V. Broude. CDK8/19 inhibition overcomes in vitro and in vivo resistance to lapatinib in HER2+ breast cancer via STAT1 and STAT3 [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 LB132.

Information design has become more and more popular these days as we have step into a smart phone new era. This document take a survey on some of the main metro station based in shanghai, and analyzed the pros and cons of the information design in existing metro system. Also explained how better information design can be put to use in order to refine user experience when taking metro.

Over 70% of breast cancer patients are estrogen receptor (ER) positive and 25% of patients over-express HER2 making these patients susceptible to therapeutic intervention with ER- and HER2-targeted therapies, respectively. However, intrinsic and acquired resistance to targeted therapies is a significant clinical issue and new therapeutic approaches aimed to preventing and overcoming resistance are urgently needed. We have previously shown that high expression of CDK8, a transcription regulating kinase, is associated with shorter relapse free survival in both ER and HER2 positive breast cancer. We have found that CDK8 inhibition by a selective small molecule inhibitor (Senexin B), by shRNA knockdown or by CRISPR/CAS9 knockout, strongly inhibits estrogen signaling in ER-positive breast cancer cells. Senexin B produces a synergistic growth inhibitory effect with an antiestrogen fulvestrant in all the tested ER-positive breast cancer cell lines in vitro and in MCF7 xenograft model in vivo. Senexin B treatment also inhibited invasive growth of MCF7 xenografts. CDK8 inhibition suppressed the emergence of estrogen independence upon long-term estrogen deprivation. A highly synergistic growth inhibitory effect occurred when Senexin B was combined with an anti-HER2 monoclonal antibody (a biosimilar of trastuzumab) or with the HER2/EGFR small molecule inhibitor lapatinib. These synergistic effects were observed in all HER2 positive breast cancer cell lines tested including those that exhibit innate and acquired resistance to HER2 targeting therapy. Furthermore, combining lapatinib with Senexin B completely abrogated the emergence of acquired lapatinib resistance. Taken together these results suggest that CDK8 inhibition, when combined with either ER- or HER2-targeted therapies, offers a rational approach to improving the efficacy of targeted drugs in breast cancer. Citation Format: McDermott MS, Chumanevich A, Liang J, Chen M, Altilia S, Hennes C, Roninson IB, Broude EV. CDK8 inhibition improves the efficacy of ER- and HER2-targeted drugs in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-05.

// Martina S.J. McDermott 1 , Alexander A. Chumanevich 1 , Chang-uk Lim 1 , Jiaxin Liang 1 , Mengqian Chen 1 , Serena Altilia 1 , David Oliver 1 , James M. Rae 2 , Michael Shtutman 1 , Hippokratis Kiaris 1 , Balazs Győrffy 3 , Igor B. Roninson 1 and Eugenia V. Broude 1 1 Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC, USA 2 University of Michigan Medical School, Ann Arbor, MI, USA 3 MTA TTK Lendulet Cancer Biomarker Research Group, Semmelweis University 2nd Department of Pediatrics, Budapest, Hungary Correspondence to: Eugenia V. Broude, email: // Keywords : CDK8, estrogen receptor, breast cancer, transcription, estrogen independence Received : January 04, 2017 Accepted : January 17, 2017 Published : January 29, 2017 Abstract Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERα expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy.

Transcription factor STAT1 is part of the JAK/STAT signaling cascade; it plays an essential role in mediating responses to interferons (IFN). STAT1 modulates many cellular processes and has been implicated in both tumor-suppressing and tumor-promoting activities. IFNγ treatment induces STAT1 phosphorylation at tyrosine 701, enabling the translocation of STAT1 from cytoplasm to the nucleus. STAT1 is also phosphorylated at serine 727; S727 phosphorylation modulates the transcriptional activity of STAT1. Transcription-regulating kinase CDK8 was shown to be primarily responsible for IFNγ-induced STAT1 S727 phosphorylation, and serine-phosphorylated form of STAT1 was proposed as a potential pharmacodynamic (PD) marker for the activity of CDK8, an emerging cancer drug target. However, the role of CDK8 in STAT1 S727 phosphorylation in the absence of IFNγ treatment is controversial, as is the role of CDK19, a closely related paralog of CDK8, as well as the relative contributions of kinase-dependent and kinase-independent activities of CDK8 and CDK19. We have found that Senexin B, a selective CDK8/19 inhibitor, decreased both IFNγ-induced and basal STAT1 S727 phosphorylation in all the tested types of tumor cells but some phosphorylation was maintained even at the highest concentrations of Senexin B. We then generated a series of derivatives of human 293 cells with CRISPR-mediated knockout of CDK8, CDK19 and both CDK8 and CDK19 (double knockout, dKO), with subsequent reconstitution of dKO with wild-type (WT) CDK8 or CDK19 or with kinase-dead (KD) mutants of these proteins. The knockout of CDK8 or CDK19 individually had only a moderate effect on IFNγ-induced and basal STAT1 S727 phosphorylation, which remained susceptible to Senexin B. In contrast, such phosphorylation was strongly decreased in dKO cells, where it was unaffected by Senexin B. Senexin B-sensitive STAT1 S727 phosphorylation was restored by re-introduction of WT CDK8 or CDK19 but not of their KD mutants. Hence, both CDK8 and CDK19 mediate STAT1 S727 phosphorylation in kinase-dependent manner. We also tested different cytokines and stress-inducing agents for the ability to induce STAT1 S727 phosphorylation in WT and dKO cells. Several treatments, including serum stimulation, EGF, TNFα, etoposide and taxol, increased this phosphorylation in dKO cells, indicating that STAT1 serine phosphorylation induced by many treatments was CDK8/19-independent. Hence, great caution needs to be utilized in using STAT1 S727 phosphorylation as a PD marker for CDK8 activity. Citation Format: Jing Li, Jiaxin Liang, Lili Wang, Eugenia Broude, Igor Roninson, Mengqian Chen. Role of CDK8 and CDK19 in STAT1 phosphorylation at serine 727 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 89.

A gene (aga0917) encoding a putative β-agarase was identified from the genome of Pseudoalteromonas fuliginea YTW1-15-1. The nucleotide sequence analysis revealed that aga0917 had significant homology to the agarase genes of the GH16 family. aga0917 encodes a putative protein of 290 amino acids with an estimated molecular mass of 32.5 kDa, including a 21-amino acid signal peptide. A gene fragment encoding only the putative mature form of Aga0917 (269 amino acids) was overexpressed in Escherichia coli BL21 (DE3) pLysS as a 6 × histine-tagged fusion protein (rmAga0917). The K

Unresectable hepatic metastases of colon cancer respond poorly to existing therapies and are a major cause of colon cancer lethality. In this study, we evaluated the therapeutic viability of targeting the mediator kinase CDK8, an early clinical stage drug target, as a means to suppress metastasis of colon cancer. CDK8 was amplified or overexpressed in many colon cancers and CDK8 expression correlated with shorter patient survival. Knockdown or inhibition of CDK8 had little effect on colon cancer cell growth but suppressed metastatic growth of mouse and human colon cancer cells in the liver. This effect was due in part to inhibition of already established hepatic metastases, indicating therapeutic potential of CDK8 inhibitors in the metastatic setting. In contrast, knockdown or inhibition of CDK8 had no significant effect on the growth of tumors implanted subcutaneously, intrasplenically, or orthotopically in the cecum. CDK8 mediated colon cancer growth in the liver through downregulation of matrix metalloproteinase (MMP) inhibitor TIMP3 via TGFβ/SMAD-driven expression of a TIMP3-targeting microRNA, miR-181b, along with induction of Mmp3 in murine or MMP9 in human colon cancer cells via Wnt/β-catenin-driven transcription. These findings reveal a new mechanism for negative regulation of gene expression by CDK8 and a site-specific role for CDK8 in colon cancer hepatic metastasis. Our results indicate the utility of CDK8 inhibitors for the treatment of colon cancer metastases in the liver and suggest that CDK8 inhibitors may be considered in other therapeutic settings involving TGFβ/SMAD or Wnt/β-catenin pathway activation. Significance: These findings demonstrate that inhibition of the transcription-regulating kinase CDK8 exerts a site-specific tumor-suppressive effect on colon cancer growth in the liver, representing a unique therapeutic opportunity for the treatment of advanced colon cancer.

CDK8 and its paralog CDK19, in complex with CCNC, MED12 and MED13, are transcriptional regulators that mediate several carcinogenic pathways and the chemotherapy-induced tumor-supporting paracrine network. Following up on our previous observation that CDK8, CDK19 and CCNC RNA expression is associated with shorter relapse-free survival (RFS) in breast cancer, we now found by immunohistochemical analysis that CDK8/19 protein is overexpressed in invasive ductal carcinomas relative to non-malignant mammary tissues. Meta-analysis of transcriptomic data revealed that higher CDK8 expression is associated with shorter RFS in all molecular subtypes of breast cancer. These correlations were much stronger in patients who underwent systemic adjuvant therapy, suggesting that CDK8 impacts the failure of systemic therapy. The same associations were found for CDK19, CCNC and MED13. In contrast, MED12 showed the opposite association with a longer RFS. The expression levels of CDK8 in breast cancer samples were directly correlated with the expression of MYC, as well as CDK19, CCNC and MED13 but inversely correlated with MED12. CDK8, CDK19 and CCNC expression was strongly increased and MED12 expression was decreased in tumors with mutant p53. Gene amplification is the most frequent type of genetic alterations of CDK8, CDK19, CCNC and MED13 in breast cancers (9.7% of which have amplified MED13), whereas point mutations are more common in MED12. These results suggest that the expression of CDK8 and its interactive genes has a profound impact on the response to adjuvant therapy in breast cancer in accordance with the role of CDK8 in chemotherapy-induced tumor-supporting paracrine activities.

CDK8, along with its paralog CDK19, is a cyclin dependent kinase which, in contrast to other members of the CDK family does not regulate cell cycle progression. CDK8 acts as a pleiotropic transcription regulator potentiating the induction of transcription by several transcription factors. Immunohistochemical staining of breast tissue arrays and bioinformatics analysis of gene expression microarray data of breast cancer patients revealed that CDK8 is overexpressed in breast cancer and that higher CDK8 expression correlates with the failure of systemic therapy. Small-molecule selective inhibitors of CDK8 and CDK19 (Senexin A and Senexin B) inhibited the mitogenic effects of estrogen and estrogen-dependent transcription in estrogen receptor (ER)+ breast cancer cell lines. CDK8/19 inhibitors had a cytostatic effect on different ER+ cell lines, and this growth inhibition was synergistic with the effect of the anti-estrogen fulvestrant, particularly in ER+ cell lines resistant to estrogen deprivation. Some of the ER+ cell lines sensitive to CDK8/19 inhibition also express HER2, and therefore we tested CDK8/19 inhibitors in combination with the HER2 and EGFR tyrosine kinase inhibitor lapatinib and an anti-HER2 monoclonal antibody, a biosimilar of trastuzumab. CDK8/19 inhibition produced a synergistic decrease in cell growth with both HER2 inhibitors; this effect was especially pronounced with a trastuzumab biosimilar. Surprisingly, the synergistic effect with HER2 inhibitors was observed in both ER+ HER2+ and ER-HER2+ cell lines, suggesting an effect on a HER2-complementing molecular target other than ER. Interestingly, CDK8/19 inhibition also synergized with trastuzumab biosimilar in a breast cancer cell line that exhibits innate resistance to trastuzumab, suggesting that CDK8/19 inhibition can overcome trastuzumab resistance in breast cancer. These results suggest that combining anti-estrogen and anti-CDK8 therapy may be more effective than conventional hormone therapy for ER positive breast cancer and that combining anti-HER2 and anti-CDK8 therapy is a rational potential treatment for HER2+ breast cancer, regardless of ER status or sensitivity to trastuzumab. Citation Format: Martina S McDermott, Chang-uk Lim, Mengqian Chen, Alexander Chumanevich, James F Catroppo, Balazs Gyorffy, David Oliver, Igor B Roninson, Eugenia V Broude. CDK8 inhibition potentiates anti-ER and anti-HER2 therapies in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-15-13.

Table S2. The sequences of primers. (DOCX 25Â kb)

Table S1. AML patientsâ characteristics. (DOCX 20Â kb)