Donald C. Porter

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.

CDK8/19 Mediator kinases potentiate induction of transcription by NFκB

Significance Nuclear factor-κB (NFκB) transcription factors have been implicated in several major diseases, including inflammatory disorders, viral infections, and cancer. NFκB-inhibiting drugs typically have side effects, possibly due to sustained NFκB suppression. The ability to affect induced, but not basal, NFκB activity could provide therapeutic benefit without associated toxicity. We report that the transcription-regulating kinases CDK8/19 potentiate NFκB activity, including the expression of tumor-promoting proinflammatory cytokines, by enabling the completion of NFκB-initiated transcription. CDK8/19 inhibitors suppress the induction of gene expression by NFκB or other transcription factors, but generally do not affect basal expression of the same genes. The role of CDK8/19 in newly induced transcription identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development, differentiation, and pathological processes. 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.

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 PR08: Targeting tumor microenvironment with selective small-molecule inhibitors of CDK8/19

Cyclin-dependent kinase 8 (CDK8), along with its closely related paralog CDK19, are transcription-regulating kinases that, unlike some other members of the CDK family, do not regulate cell cycle progression and are not required for the growth of normal cells or most of the tumor cell types. CDK8 has been identified as an oncogene that enhances the activity of several tumor-promoting transcriptional pathways (such as TGFβ, β-catenin, HIF1A and serum factors), mediating the elongation of transcription of newly activated genes. We have previously reported the discovery of the first selective small-molecule inhibitors of CDK8/19 and their ability to block chemotherapy-induced tumor-promoting paracrine activities of both tumor and normal cells (Porter et al., PNAS 109, 13799, 2012). We now conducted chemical optimization of the original inhibitors, yielding an optimized preclinical lead compound, Senexin B. Senexin B inhibits CDK8/19 in low nanomolar range in vitro and in vivo as an ATP pocket binder, with very high target selectivity as indicated by kinome profiling. It is highly water-soluble, bioavailable, and produces no limiting toxicity upon prolonged administration in mice, at doses that yield plasma concentrations exceeding cellular IC50 by 2-3 orders of magnitude. Senexin B has been tested for efficacy in several animal models addressing different aspects of tumor growth and progression. (i) Pretreatment of tumor-free mice with Senexin B significantly inhibited the growth of triple-negative breast cancer (TNBC) cells inoculated into mice subsequently to Senexin B administration, indicating a general chemopreventive effect on the normal tissue “soil”. (ii) Senexin B potentiated the tumor-suppressive effect of doxorubicin on established TNBC xenografts; this effect was associated with the suppression of NFκB-mediated transcriptional induction of tumor-promoting cytokines. (iii) Senexin B inhibited invasive growth into the muscle layer in an orthotopic xenograft model of MDA-MB-468 TNBC cells. (iv) In a spleen-to-liver colon cancer metastasis model of syngeneic mouse CT26 tumors, Senexin B treatment of mice had the same effect as CDK8 knockdown in tumor cells: suppression of metastatic growth in the liver without a significant effect on primary tumor growth in the spleen. Taken together, these results indicate that CDK8/19 inhibition produces chemopotentiating, chemopreventive and anti-metastatic effects in different types of cancer, inhibiting tumor progression by acting both at the tumor cells (the “seed”) and the tumor microenvironment (the “soil”) of cancers. Citation Format: Donald C. Porter, Mengqian Chen, Jiaxin Liang, Vimala Kaza, Alexander Chumanevich, Serena Altilia, Elena Farmaki, Marj Pena, Gary P. Schools, Ioulia Chatzistamou, Lawrence T. Friedhoff, Mark P. Wentland, Eugenia V. Broude, Hippokratis Kiaris, Igor B. Roninson. Targeting tumor microenvironment with selective small-molecule inhibitors of CDK8/19. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR08. doi:10.1158/1538-7445.CHTME14-PR08

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

Abstract 4879: Targeting the seed and the soil of cancers with selective small-molecule inhibitors of CDK8/19: Chemopotentiating, chemopreventive, anti-invasive and anti-metastatic activities

Cyclin-dependent kinase 8 (CDK8), along with its closely related paralog CDK19, are transcription-regulating kinases that, unlike some other members of the CDK family, do not regulate cell cycle progression and are not required for the growth of normal cells or most of the tumor cell types. CDK8 has been identified as an oncogene that enhances the activity of several tumor-promoting transcription factors, mediating the elongation of transcription of newly activated genes. We have previously reported the discovery of the first selective small-molecule inhibitors of CDK8/19 and their ability to block chemotherapy-induced tumor-promoting paracrine activities of both tumor and normal cells (Porter et al., PNAS 109, 13799, 2012). We now conducted chemical optimization of the original inhibitors, yielding an optimized preclinical lead compound, Senexin B. Senexin B inhibits CDK8/19 in low nanomolar range in vitro and in vivo as an ATP pocket binder, with very high target selectivity as indicated by kinome profiling. It is highly water-soluble, bioavailable, and produces no limiting toxicity upon prolonged administration in mice, at doses that yield plasma concentrations exceeding cellular IC50 by 2-3 orders of magnitude. Senexin B has been tested for efficacy in several animal models addressing different aspects of tumor growth and progression. (i) Pretreatment of tumor-free mice with Senexin B significantly inhibited the growth of triple-negative breast cancer (TNBC) cells inoculated into mice subsequently to Senexin B administration, indicating a general chemopreventive effect on the normal tissue “soil”. (ii) Senexin B potentiated the tumor-suppressive effect of doxorubicin on established TNBC xenografts. (iii) Senexin B inhibited invasive growth into the muscle layer in an orthotopic xenograft model of MDA-MB-468 TNBC cells. (iv) In a spleen-to-liver metastasis model of syngeneic mouse CT26 tumors, Senexin B treatment of mice had the same effect as CDK8 knockdown in tumor cells: suppression of metastatic growth in the liver without a significant effect on primary tumor growth in the spleen. Taken together, these results indicate that CDK8/19 inhibition produces chemopotentiating, chemopreventive and anti-metastatic effects in different types of cancer, inhibiting tumor progression at the organismal level. Citation Format: Donald C. Porter, Jiaxin Liang, Vimala Kaza, Alexander A. Chumanevich, Serena Altilia, Elena Farmaki, Mengqian Chen, Gary P. Schools, Ioulia Chatzistamou, Marj M. Pena, Lawrence T. Friedhoff, Mark P. Wentland, Eugenia Broude, Hippokratis Kiaris, Igor B. Roninson. Targeting the seed and the soil of cancers with selective small-molecule inhibitors of CDK8/19: Chemopotentiating, chemopreventive, anti-invasive and anti-metastatic activities. [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 4879. doi:10.1158/1538-7445.AM2014-4879

Abstract 1820: CDK3: A novel tumor-selective drug target involved in AP1 activation and transcriptional damage response

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cellular damage by chemotherapy or radiation induces transcriptional damage response that leads to increased production of tumor-promoting cytokines and decreases the treatment efficacy. The same response can be triggered by the overexpression of a damage-inducible cell cycle inhibitor p21 (Chang et al., PNAS 97, 4291, 2000 and PNAS 99, 389, 2002). High-throughput screening of diversified small-molecule libraries for the ability to prevent the induction of transcription in p21-expressing cells yielded a number of inhibitors of this transcriptional response. One of these compounds, designated SNX9, and a series of SNX9-related molecules, also showed unexpected cytotoxicity that was selective for tumor relative to normal cells. SNX9-class compounds strongly inhibited transcriptional activation of different tumor-promoting cytokines in irradiated colon cancer cells and potentiated the induction of apoptosis by doxorubicin and irinotecan. SNX9-class compounds showed tumoricidal activity against different tumor cell types in vitro and against colon carcinoma xenografts in vivo. Kinome profiling, followed by siRNA verification, identified the target of SNX9-class compounds as CDK3, a member of cyclin-dependent kinase (CDK) family. While CDK3 is closely related to cell cycle regulating kinase CDK2, it is not required for cell cycle progression in normal cells, judging by its spontaneous mutational inactivation in the germline of laboratory mice (Ye et al., PNAS 98, 1682, 2001) and very low expression in human tissues. CDK3, however, was reported to be overexpressed in tumor cells, where it displays oncogenic activity due to its ability to activate AP1 (Jun/Fos), a transcription factor involved in carcinogenesis (Zheng et al., Cancer Res., 68, 7650, 2008; Cho et al., Cancer Res., 69, 272, 2009). In agreement with these findings, AP1 was upregulated upon p21 expression and inhibited by SNX9-class CDK3 inhibitors. SNX9-class compounds inhibited CDK3 preferentially to CDK2 and other CDKs, despite the fact that the ATP pocket, the usual active site of kinase inhibitors, is highly conserved between CDK3 and CDK2. ATP competition assays revealed that SNX9-class compounds do not act at the ATP pocket, and molecular modeling suggested a novel allosteric binding pocket for these compounds, which is distinct between CDK3 and CDK2. Tumor-selective expression and function of CDK3 and its roles in AP1 activation, tumor cell survival and transcriptional damage response suggest this kinase as an exceptionally promising new target for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1820. doi:1538-7445.AM2012-1820