Chang-uk Lim

Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities

Conventional chemotherapy not only kills tumor cells but also changes gene expression in treatment-damaged tissues, inducing production of multiple tumor-supporting secreted factors. This secretory phenotype was found here to be mediated in part by a damage-inducible cell-cycle inhibitor p21 (CDKN1A). We developed small-molecule compounds that inhibit damage-induced transcription downstream of p21. These compounds were identified as selective inhibitors of a transcription-regulating kinase CDK8 and its isoform CDK19. Remarkably, p21 was found to bind to CDK8 and stimulate its kinase activity. p21 and CDK8 also cooperate in the formation of internucleolar bodies, where both proteins accumulate. A CDK8 inhibitor suppresses damage-induced tumor-promoting paracrine activities of tumor cells and normal fibroblasts and reverses the increase in tumor engraftment and serum mitogenic activity in mice pretreated with a chemotherapeutic drug. The inhibitor also increases the efficacy of chemotherapy against xenografts formed by tumor cell/fibroblast mixtures. Microarray data analysis revealed striking correlations between CDK8 expression and poor survival in breast and ovarian cancers. CDK8 inhibition offers a promising approach to increasing the efficacy of cancer chemotherapy.

Networks of intergenic long-range enhancers and snpRNAs drive castration-resistant phenotype of prostate cancer and contribute to pathogenesis of multiple common human disorders.

The mechanistic relevance of intergenic disease-associated genetic loci (IDAGL) containing highly statistically significant disease-linked SNPs remains unknown. Here, we present experimental and clinical evidence supporting the importantance of the role of IDAGL in human diseases. A targeted RT-PCR screen coupled with sequencing of purified PCR products detects widespread transcription at multiple IDAGL and identifies 96 small noncoding trans-regulatory RNAs of ~100–300 nt in length containing SNPs (snpRNAs) associated with 21 common disorders. Multiple independent lines of experimental evidence support functionality of snpRNAs by documenting their cell type-specific expression and evolutionary conservation of sequences, genomic coordinates and biological effects. Chromatin state signatures, expression profiling experiments and luciferase reporter assays demonstrate that many IDAGL are Polycomb-regulated long-range enhancers. Expression of snpRNAs in human and mouse cells markedly affects cellular behavior and induces allele-specific clinically relevant phenotypic changes: NLRP1-locus snpRNAs rs2670660 exert regulatory effects on monocyte/macrophage transdifferentiation, induce prostate cancer (PC) susceptibility snpRNAs and transform low-malignancy hormone-dependent human PC cells into highly malignant androgen-independent PC. Q-PCR analysis and luciferase reporter assays demonstrate that snpRNA sequences represent allele-specific “decoy” targets of microRNAs that function as SNP allele-specific modifiers of microRNA expression and activity. We demonstrate that trans-acting RNA molecules facilitating resistance to androgen depletion (RAD) in vitro and castration-resistant phenotype (CRP) in vivo of PC contain intergenic 8q24-locus SNP variants (rs1447295; rs16901979; rs6983267) that were recently linked with increased risk of PC. Q-PCR analysis of clinical samples reveals markedly increased and highly concordant (r = 0.896; p < 0.0001) snpRNA expression levels in tumor tissues compared with the adjacent normal prostate [122-fold and 45-fold in Gleason 7 tumors (p = 0.03); 370-fold and 127-fold in Gleason 8 tumors (p = 0.0001) for NLRP1-locus and 8q24-locus snpRNAs, respectively]. Our experiments indicate that RAD and CR phenotype of human PC cells can be triggered by ncRNA molecules transcribed from the NLRP1-locus intergenic enhancer at 17p13 and by downstream activation of the 8q24-locus snpRNAs. Our results define the IDAGL at 17p13 and 8q24 as candidate regulatory loci of RAD and CR phenotypes of PC, reveal previously unknown molecular links between the innate immunity/inflammasome system and development of hormone-independent PC and identify novel molecular and genetic targets with diagnostic and therapeutic potentials, exploration of which should be highly beneficial for personalized clinical management of PC.

Tumor-specific silencing of COPZ2 gene encoding coatomer protein complex subunit ζ2 renders tumor cells dependent on its paralogous gene COPZ1

Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the coatomer protein complex ζ1 (COPZ1) gene as essential for different tumor cell types but not for normal cells. COPZ1 encodes a subunit of coatomer protein complex 1 (COPI) involved in intracellular traffic and autophagy. The knockdown of COPZ1, but not of COPZ2 encoding isoform coatomer protein complex ζ2, caused Golgi apparatus collapse, blocked autophagy, and induced apoptosis in both proliferating and nondividing tumor cells. In contrast, inhibition of normal cell growth required simultaneous knockdown of both COPZ1 and COPZ2. COPZ2 (but not COPZ1) was down-regulated in the majority of tumor cell lines and in clinical samples of different cancer types. Reexpression of COPZ2 protected tumor cells from killing by COPZ1 knockdown, indicating that tumor cell dependence on COPZ1 is the result of COPZ2 silencing. COPZ2 displays no tumor-suppressive activities, but it harbors microRNA 152, which is silenced in tumor cells concurrently with COPZ2 and acts as a tumor suppressor in vitro and in vivo. Silencing of microRNA 152 in different cancers and the ensuing down-regulation of its host gene COPZ2 offer a therapeutic opportunity for proliferation-independent selective killing of tumor cells by COPZ1-targeting agents.

Effects of conditional depletion of topoisomerase II on cell cycle progression in mammalian cells

Topoisomerase II (Topo II) that decatenates newly synthesized DNA is targeted by many anticancer drugs. Some of these drugs stabilize intermediate complexes of DNA with Topo II and others act as catalytic inhibitors of Topo II. Simultaneous depletion of Topo IIα and Topo IIβ, the two isoforms of mammalian Topo II, prevents cell growth and normal mitosis, but the role of Topo II in other phases of mammalian cell cycle has not yet been elucidated. We have developed a derivative of p53-suppressed human cells with constitutive depletion of Topo IIβ and doxycycline-regulated conditional depletion of Topo IIα. The effects of Topo II depletion on cell cycle progression were analyzed by time-lapse video microscopy, pulse-chase flow cytometry and mitotic morphology. Topo II depletion increased the duration of the cell cycle and mitosis, interfered with chromosome condensation and sister chromatid segregation and led to frequent failure of cell division, ending in either cell death or restitution of polyploid cells. Topo II depletion did not change the rate of DNA replication but increased the duration of G2. These results define the effects of decreased Topo II activity, rather than intermediate complex stabilization, on the mammalian cell cycle.

Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer

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.

Abstract 1512: Functional characterization of novel transcription-regulating cancer drug targets, CDK8 and CDK19, using CRISPR/Cas9 knockout and a highly selective CDK8/19 kinase inhibitor

The Mediator complex-associated cyclin-dependent kinase CDK8 is an oncogenic transcription-regulating serine/threonine kinase that mediates multiple cancer-associated transcriptional pathways. Despite recent high-profile attention to CDK8 as a novel cancer drug target, very little is known about the function of CDK8’s closely related paralog CDK19. Using CRISPR/Cas9n system we generated CDK8/CDK19 single-knockout (CDK8-KO and CDK19-KO) and double-knockout (CDK8/19-dKO) derivatives of HEK293 cells. RNA-Seq was used to characterize the effects of a highly selective small-molecule CDK8/19 kinase inhibitor Senexin B on gene expression in the parental, single-knockout and double-knockout cell lines. This analysis was conducted in the absence or in the presence of TNF-alpha, an inducer of transcription factor NF-kappa-B that we have previously shown to be potentiated by CDK8. The following results were obtained. (1) CDK8 and CDK19 have complementary functions in stabilization of their partner Cyclin C (independent of their kinase activity) and phosphorylation of transcription factor STAT1 at S727 (dependent on the kinase activity). (2) Senexin B treatment affected gene expression in wild-type 293 cells but had almost no effect in CDK8/19-dKO cells, indicating a very high degree of target selectivity. (3) In contrast to the results with CDK8/19-dKO, most of the genes affected by Senexin B in the wild-type cells were also affected in CDK8-KO and CDK19-KO cells, indicating complementary functions of CDK8 and CDK19. (4) Re-expression of either CDK8 or CDK19, but not of a CDK8 kinase-dead (D173A) mutant, in CDK8/19-dKO cells restored CDK8/19 kinase-dependent gene expression as well as the regulatory effects of Senexin B. (5) Many more genes were inhibited rather than induced by Senexin B, indicating that CDK8/19 act primarily as positive regulators of transcription. (6) The total number of genes affected by Senexin B was greatly increased in cells treated with TNF-alpha, suggesting that cooperation with other transcription factors (such as NF-kappa-B) is the primary role of CDK8/19. Our results indicate that complete suppression of cancer-relevant activities of CDK8 requires simultaneous inhibition of both CDK8 and CDK19. Citation Format: Mengqian Chen, Bing Hu, Hao Ji, Serena Altilia, Jiaxin Liang, Martina McDermott, Chang-uk Lim, Donald C. Porter, Eugenia Broude, Igor Roninson. Functional characterization of novel transcription-regulating cancer drug targets, CDK8 and CDK19, using CRISPR/Cas9 knockout and a highly selective CDK8/19 kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1512. doi:10.1158/1538-7445.AM2017-1512

Abstract 4896: Role of CDK8 in colon cancer hepatic metastasis

About half of all colon cancer patients will develop liver metastases and the 5-year survival for these patients is less than 13%, making colon cancer the second most lethal cancer worldwide. Cyclin dependent kinase 8 (CDK8), which regulates transcription but not cell cycle progression, has been identified as an oncogene amplified in many colon cancers. CDK8 acts as a positive mediator of oncogenic transcription pathways regulated by Wnt/beta-catenin and TGF-beta, both of which are strongly associated with tumor metastasis. In a colon cancer liver metastasis model based on splenic injection of CT26 murine colon carcinoma cells, treatment with Senexin B, a highly selective small-molecule inhibitor of CDK8 and its paralog CDK19, strongly inhibited metastatic growth in the liver and prolonged the survival of mice with hepatic metastases. In contrast to the effect on liver metastasis, CDK8 inhibition had little or no effect on cell growth in culture or at primary tumor sites. Hepatic metastasis was inhibited to the same extent when Senexin B was administered starting from the time of tumor injection or only during a later part of the study, suggesting that the drug affected metastatic growth in the liver rather than just initial colonization. Liver metastasis was similarly decreased by CDK8 knockdown in CT26 cells, suggesting that the anti-metastatic activity of Senexin B was due at least in part to its effect on tumor cells. Transcription profiling indicated that CDK8 inhibition by shRNA or Senexin B strongly decreased the expression of metastasis-associated metalloproteinases MMP 13, MMP10, and MMP3 and at the same time drastically increased the expression of TIMP3, a metalloproteinase inhibitor. TIMP3 overexpression in CT26 cells, like CDK8 inhibition, decreased hepatic metastasis. CDK8 inhibition also blocked TGF-beta or Wnt3a-stimulated transwell Matrigel invasion of CT26 cells in vitro. Knockdown of beta-catenin in CT26 cells decreased the expression of MMP3 and MMP13, while knockdown of SMAD4 (transcriptional mediator of TGF-beta pathway) induced TIMP3 expression. Beta-catenin knockdown decreased both the primary tumor growth in the spleen and metastatic growth in the liver, whereas SMAD4 knockdown, like that of CDK8, selectively inhibited liver metastasis. Senexin B also suppressed hepatic metastasis after splenic injection of human HCT116 colon carcinoma cells. In summary, our data identified CDK8 as a key transcriptional regulator of colon cancer metastatic growth in the liver, interacting with TGF-beta and Wnt/beta-catenin pathways and regulating the expression of MMPs and Timp3. CDK8/19 inhibitors, which are now entering clinical trials, may be effective for the treatment of hepatic metastasis of colon cancer. Citation Format: Jiaxin Liang, Mengqian Chen, Mythreye Karthikeyan, M. Marjorette Pena, Daniel Hughes, Vimala Kaza, Chang-Uk Lim, Eugenia Broude, Igor B. Roninson. Role of CDK8 in colon cancer hepatic metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4896. doi:10.1158/1538-7445.AM2017-4896

Abstract P4-15-13: CDK8 inhibition potentiates anti-ER and anti-HER2 therapies in breast cancer

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.

Abstract 2101: Role of CDK8 in estrogen receptor signaling in breast cancers

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The majority of breast cancers (BrCa) overexpress estrogen receptor (ER)α, which regulates transcription and drives estrogen-stimulated proliferation of ER+ tumor cells. ER+ patients usually receive adjuvant anti-estrogen therapy based on ER modification, downregulation, or estrogen depletion. Tumors frequently develop resistance to anti-estrogen therapy through various mechanisms, which may involve stimulation of ER itself or of downstream mediators of ER-driven transcription, as well as activation of alternative proliferation pathways, in particular those driven by HER2/EGFR. The treatment efficacy of hormone-resistant BrCa could be greatly augmented by targeting new druggable mediators of ER activity. We have now identified a transcription-regulating oncogenic kinase CDK8 as a potentiator of ER signaling and ER-driven BrCa cell proliferation. Immunohistochemical staining of breast tissue arrays and bioinformatics analysis of gene expression microarray data of breast cancer patients revealed that CDK8 is overexpressed in BrCa and that higher CDK8 expression correlates with the failure of systemic therapy. Among systemically untreated patients, higher CDK8 expression was correlated with shorter relapse-free survival in a subset of ER+ tumors that expressed the lowest levels of ERα. This correlation suggested that CDK8 could play a role in the progression of ER+ breast cancers with reduced levels of ER, possibly by stimulating the mitogenic effects of ER-mediated transcription. Indeed, a selective small-molecule CDK8 inhibitor (Senexin A) decreased estrogen-induced ER-dependent transcription and inhibited estrogen-stimulated proliferation of ER+ BrCa cell lines. Microarray analysis revealed that CDK8 inhibition diminished the induction of genes that show rapid and sustained activation by estrogen in ER+ cells. CDK8 inhibition had an additive effect in combination with anti-estrogens in ER+ BrCa and a synergistic effect with fulvestrant in BrCa cells resistant to estrogen deprivation. Some of these cell lines also express HER2/EGFR, and CDK8 inhibition in combination with a HER2/EGFR inhibitor lapatinib synergistically inhibited the growth of these cells. These results suggest that combining anti-estrogen and anti-CDK8 therapy may be more effective than conventional hormone therapy for ER+ BrCa. Citation Format: Martina McDermott, Balazs Gyorffy, Chang-uk Lim, Alexander Chumanevich, Zhengguan Yang, Mengqian Chen, James F. Catroppo, Igor Roninson, Eugenia V. Broude. Role of CDK8 in estrogen receptor signaling in breast cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2101. doi:10.1158/1538-7445.AM2014-2101

Abstract 4883: CDK8: A new druggable mediator of NFκB activity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cyclin-dependent kinase 8 (CDK8) is a transcription-regulating oncogenic serine kinase, which mediates several cancer-related transcriptional pathways such as beta-catenin, TP53, TGF-beta and HIF1A. We have recently discovered the first selective small-molecule inhibitors of CDK8 and its closely related isoform CDK19 and showed that these inhibitors block chemotherapy-induced tumor-promoting paracrine activities of both tumor and normal cells (Porter et al., PNAS 109, 13799, 2012). Since transcription factor NFκB plays a key role in damage-induced expression of tumor-promoting cytokines, we have tested the effects of a highly selective CDK8/19 inhibitor Senexin A on NFκB-induced transcription. Senexin A treatment or knockdown of CDK8 by shRNA inhibits transcriptional activation of NFκB-responsive promoters and of acutely responsive cytokine genes (such as CXCL1, CXCL2, IL8 and CCL20), by TNFα, a canonical NFκB activator, in several cell lines. CDK8 inhibition did not prevent nuclear translocation of active NFκB proteins. Chromatin immunoprecipitation (ChIP) analysis of HEK293 cells, untreated or treated with TNFα or Senexin A, singly or in combination, showed that CDK8 is recruited to NFκB early-responsive genes upon TNFα treatment, but the CDK8 inhibitor did not prevent the recruitment of NFκB, CDK8 or RNA Polymerase II (Pol II) to the target genes. However, CDK8 kinase activity was found to be required for C-terminal domain phosphorylation (both at S2 and S5 residues) of Pol II associated with the target genes, which is needed for the elongation of their NFκB-initiated transcription. In contrast, Pol II phosphorylation at constitutively expressed genes is not dependent on CDK8 kinase activity. These results suggest that CDK8 inhibitors may exert their effects against the activation of tumor-promoting and pro-inflammatory secreted factors in the tumor microenvironment by preventing transcriptional activation of NFκB-induced genes. Citation Format: Mengqian Chen, Martina McDermott, Zhengguan Yang, Jiaxin Liang, Gary P. Schools, Chang-uk Lim, Tao Lu, Stark R. George, Deborah K. West, Donald C. Porter, Eugenia V. Broude, Igor B. Roninson. CDK8: A new druggable mediator of NFκB activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4883. doi:10.1158/1538-7445.AM2014-4883