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• W2170846478 abstract "•THZ1, a covalent CDK7 inhibitor, is highly potent against MYC-driven cancer cells•THZ1 causes MYCN-amplified neuroblastoma regression without toxicity•THZ1 acts by suppressing MYCN-induced global transcriptional amplification•THZ1 selectivity correlates with downregulation of super-enhancer-associated genes The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins. The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins. Many human cancers depend on the deregulated expression of MYC family members for their aberrant growth and proliferation, with elevated expression of these oncogenes predicting aggressive disease and a poor clinical outcome (Eilers and Eisenman, 2008Eilers M. Eisenman R.N. Myc’s broad reach.Genes Dev. 2008; 22: 2755-2766Crossref PubMed Scopus (739) Google Scholar, Wasylishen and Penn, 2010Wasylishen A.R. Penn L.Z. Myc: the beauty and the beast.Genes Cancer. 2010; 1: 532-541Crossref PubMed Scopus (47) Google Scholar). Deactivation of MYC in cell lines and MYC-induced transgenic tumors causes proliferative arrest and tumor regression (Arvanitis and Felsher, 2006Arvanitis C. Felsher D.W. Conditional transgenic models define how MYC initiates and maintains tumorigenesis.Semin. Cancer Biol. 2006; 16: 313-317Crossref PubMed Scopus (123) Google Scholar, Soucek et al., 2008Soucek L. Whitfield J. Martins C.P. Finch A.J. Murphy D.J. Sodir N.M. Karnezis A.N. Swigart L.B. Nasi S. Evan G.I. Modelling Myc inhibition as a cancer therapy.Nature. 2008; 455: 679-683Crossref PubMed Scopus (609) Google Scholar), suggesting that effective targeting of MYC proteins would have broad therapeutic benefit. Recently, several groups reported that oncogenic MYC elicits its plethora of downstream effects in tumor cells through global transcriptional amplification, leading to massively upregulated expression of genes involved in multiple processes (Lin et al., 2012Lin C.Y. Lovén J. Rahl P.B. Paranal R.M. Burge C.B. Bradner J.E. Lee T.I. Young R.A. Transcriptional amplification in tumor cells with elevated c-Myc.Cell. 2012; 151: 56-67Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, Lovén et al., 2012Lovén J. Orlando D.A. Sigova A.A. Lin C.Y. Rahl P.B. Burge C.B. Levens D.L. Lee T.I. Young R.A. Revisiting global gene expression analysis.Cell. 2012; 151: 476-482Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, Nie et al., 2012Nie Z. Hu G. Wei G. Cui K. Yamane A. Resch W. Wang R. Green D.R. Tessarollo L. Casellas R. et al.c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells.Cell. 2012; 151: 68-79Abstract Full Text Full Text PDF PubMed Google Scholar, Schuhmacher and Eick, 2013Schuhmacher M. Eick D. Dose-dependent regulation of target gene expression and cell proliferation by c-Myc levels.Transcription. 2013; 4: 192-197Crossref PubMed Google Scholar). When present at physiological levels, MYC binds to the core promoters of actively transcribed genes; however, in tumor cells with MYC overexpression, increased MYC levels are observed at both the core promoters and enhancers of the same set of genes, resulting in increased levels of transcripts per cell. This mechanism provides an explanation for the lack of a common MYC transcriptional signature and for the diverse effects of deregulated MYC in cancer cells. Another general feature of deregulated MYC is its transcriptional regulation by super-enhancers (SEs), clusters of enhancers that are densely occupied by transcription factors, cofactors, and chromatin regulators (Hnisz et al., 2013Hnisz D. Abraham B.J. Lee T.I. Lau A. Saint-André V. Sigova A.A. Hoke H.A. Young R.A. Super-enhancers in the control of cell identity and disease.Cell. 2013; 155: 934-947Abstract Full Text Full Text PDF PubMed Scopus (2102) Google Scholar). SEs are acquired by cancer cells through gene amplification, translocation or transcription factor overexpression. They facilitate high-level expression of genes, including MYC, whose protein products are critical for the control of cell identity, growth, and proliferation, and which are especially sensitive to perturbation (Chapuy et al., 2013Chapuy B. McKeown M.R. Lin C.Y. Monti S. Roemer M.G. Qi J. Rahl P.B. Sun H.H. Yeda K.T. Doench J.G. et al.Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma.Cancer Cell. 2013; 24: 777-790Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, Hnisz et al., 2013Hnisz D. Abraham B.J. Lee T.I. Lau A. Saint-André V. Sigova A.A. Hoke H.A. Young R.A. Super-enhancers in the control of cell identity and disease.Cell. 2013; 155: 934-947Abstract Full Text Full Text PDF PubMed Scopus (2102) Google Scholar, Lovén et al., 2013Lovén J. Hoke H.A. Lin C.Y. Lau A. Orlando D.A. Vakoc C.R. Bradner J.E. Lee T.I. Young R.A. Selective inhibition of tumor oncogenes by disruption of super-enhancers.Cell. 2013; 153: 320-334Abstract Full Text Full Text PDF PubMed Scopus (1814) Google Scholar). These emerging insights into the role of oncogenic MYC as an SE-associated transcriptional amplifier suggest that strategies aimed at disrupting the molecular mechanisms that drive this function might provide useful therapy for different MYC-dependent tumors. The transcription cycle of RNA polymerase II (Pol II) is regulated by a set of cyclin-dependent kinases (CDKs) that have critical roles in transcription initiation and elongation (Larochelle et al., 2012Larochelle S. Amat R. Glover-Cutter K. Sansó M. Zhang C. Allen J.J. Shokat K.M. Bentley D.L. Fisher R.P. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.Nat. Struct. Mol. Biol. 2012; 19: 1108-1115Crossref PubMed Scopus (259) Google Scholar). In contrast to the cell-cycle CDKs which are largely responsible for cell-cycle transition, these transcriptional CDKs (especially CDK7, a subunit of TFIIH, and CDK9, a subunit of pTEFb) phosphorylate the carboxy-terminal domain (CTD) of Pol II, facilitating efficient transcriptional initiation, pause release and elongation. Moreover, most CDKs are activated through T-loop phosphorylation by a CDK-activating kinase (CAK), which in metazoans appears to be uniquely controlled by CDK7 (Fisher and Morgan, 1994Fisher R.P. Morgan D.O. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase.Cell. 1994; 78: 713-724Abstract Full Text PDF PubMed Scopus (557) Google Scholar, Glover-Cutter et al., 2009Glover-Cutter K. Larochelle S. Erickson B. Zhang C. Shokat K. Fisher R.P. Bentley D.L. TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II.Mol. Cell. Biol. 2009; 29: 5455-5464Crossref PubMed Scopus (224) Google Scholar, Larochelle et al., 2007Larochelle S. Merrick K.A. Terret M.E. Wohlbold L. Barboza N.M. Zhang C. Shokat K.M. Jallepalli P.V. Fisher R.P. Requirements for Cdk7 in the assembly of Cdk1/cyclin B and activation of Cdk2 revealed by chemical genetics in human cells.Mol. Cell. 2007; 25: 839-850Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, Larochelle et al., 2012Larochelle S. Amat R. Glover-Cutter K. Sansó M. Zhang C. Allen J.J. Shokat K.M. Bentley D.L. Fisher R.P. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.Nat. Struct. Mol. Biol. 2012; 19: 1108-1115Crossref PubMed Scopus (259) Google Scholar, Rossignol et al., 1997Rossignol M. Kolb-Cheynel I. Egly J.M. Substrate specificity of the cdk-activating kinase (CAK) is altered upon association with TFIIH.EMBO J. 1997; 16: 1628-1637Crossref PubMed Scopus (168) Google Scholar, Serizawa et al., 1995Serizawa H. Mäkelä T.P. Conaway J.W. Conaway R.C. Weinberg R.A. Young R.A. Association of Cdk-activating kinase subunits with transcription factor TFIIH.Nature. 1995; 374: 280-282Crossref PubMed Google Scholar). Inhibition of transcriptional CDKs primarily affects the accumulation of transcripts with short half-lives, including antiapoptosis family members and cell-cycle regulators (Garriga and Graña, 2004Garriga J. Graña X. Cellular control of gene expression by T-type cyclin/CDK9 complexes.Gene. 2004; 337: 15-23Crossref PubMed Scopus (142) Google Scholar, Lam et al., 2001Lam L.T. Pickeral O.K. Peng A.C. Rosenwald A. Hurt E.M. Giltnane J.M. Averett L.M. Zhao H. Davis R.E. Sathyamoorthy M. et al.Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.Genome Biol. 2001; 2 (RESEARCH0041)Crossref Google Scholar), rendering this group of kinases ideal candidates for blocking MYC-dependent transcriptional amplification. Here, we investigate whether inhibition of transcriptional CDKs can be exploited to disrupt aberrant MYC-driven transcription, using the deregulated expression of MYCN as a model. The MYCN protein shares most of the physical properties of MYC (Kohl et al., 1986Kohl N.E. Legouy E. DePinho R.A. Nisen P.D. Smith R.K. Gee C.E. Alt F.W. Human N-myc is closely related in organization and nucleotide sequence to c-myc.Nature. 1986; 319: 73-77Crossref PubMed Scopus (161) Google Scholar) and is considered functionally interchangeable, based on the similarity of their transcriptional programs, the cellular phenotypes they induce, and the ability of MYCN to replace MYC during murine development (Boon et al., 2001Boon K. Caron H.N. van Asperen R. Valentijn L. Hermus M.C. van Sluis P. Roobeek I. Weis I. Voûte P.A. Schwab M. Versteeg R. N-myc enhances the expression of a large set of genes functioning in ribosome biogenesis and protein synthesis.EMBO J. 2001; 20: 1383-1393Crossref PubMed Scopus (341) Google Scholar, Malynn et al., 2000Malynn B.A. de Alboran I.M. O’Hagan R.C. Bronson R. Davidson L. DePinho R.A. Alt F.W. N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation.Genes Dev. 2000; 14: 1390-1399Crossref PubMed Google Scholar, Toyoshima et al., 2012Toyoshima M. Howie H.L. Imakura M. Walsh R.M. Annis J.E. Chang A.N. Frazier J. Chau B.N. Loboda A. Linsley P.S. et al.Functional genomics identifies therapeutic targets for MYC-driven cancer.Proc. Natl. Acad. Sci. USA. 2012; 109: 9545-9550Crossref PubMed Scopus (189) Google Scholar). In neuroblastoma (NB), a pediatric solid tumor arising in the peripheral sympathetic nervous system, MYCN amplification is typically associated with a dismal prognosis, regardless of the treatment used (Brodeur et al., 1984Brodeur G.M. Seeger R.C. Schwab M. Varmus H.E. Bishop J.M. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage.Science. 1984; 224: 1121-1124Crossref PubMed Scopus (1818) Google Scholar, Seeger et al., 1985Seeger R.C. Brodeur G.M. Sather H. Dalton A. Siegel S.E. Wong K.Y. Hammond D. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas.N. Engl. J. Med. 1985; 313: 1111-1116Crossref PubMed Google Scholar). We demonstrate that THZ1, a newly developed covalent inhibitor of CDK7 (Kwiatkowski et al., 2014Kwiatkowski N. Zhang T. Rahl P.B. Abraham B.J. Reddy J. Ficarro S.B. Dastur A. Amzallag A. Ramaswamy S. Tesar B. et al.Targeting transcription regulation in cancer with a covalent CDK7 inhibitor.Nature. 2014; 511: 616-620Crossref PubMed Scopus (536) Google Scholar), selectively targets MYCN-amplified NB cells, leading to global repression of MYCN-dependent transcriptional amplification. This response induces sustained growth inhibition of tumors in a mouse model of NB. The remarkable sensitivity of MYCN-amplified cells to CDK7 inhibition is associated with preferentially reduced expression of SE-associated oncogenic drivers, especially MYCN. To identify CDKs whose depletion might lead to decreased MYCN expression and subsequent apoptosis, we performed a short hairpin (shRNA) knockdown screen of CDKs with known transcriptional activities (CDK7, CDK8, CDK9, CDK12, CDK13, and CDK19) in Kelly cells, a human NB line that expresses very high levels of MYCN RNA and protein due to genomically amplified MYCN (100–120 copies per cell) (Schwab et al., 1983Schwab M. Alitalo K. Klempnauer K.H. Varmus H.E. Bishop J.M. Gilbert F. Brodeur G. Goldstein M. Trent J. Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour.Nature. 1983; 305: 245-248Crossref PubMed Scopus (1045) Google Scholar). Genetic depletion of CDK7, CDK8, CDK9, or CDK19 led to marked decreases of MYCN RNA and protein with a concomitant increase in cleaved caspase 3 (CC3) expression (Figures S1A and S1B available online). To reproduce these results pharmacologically, we tested a panel of 11 inhibitors with activity against transcriptional CDKs in three MYCN-amplified cell lines, observing a range of sensitivities, with the highest potency (IC50, 6–9 nM) shown by a newly developed covalent phenylaminopyrimidine inhibitor of CDK7, THZ1 (Kwiatkowski et al., 2014Kwiatkowski N. Zhang T. Rahl P.B. Abraham B.J. Reddy J. Ficarro S.B. Dastur A. Amzallag A. Ramaswamy S. Tesar B. et al.Targeting transcription regulation in cancer with a covalent CDK7 inhibitor.Nature. 2014; 511: 616-620Crossref PubMed Scopus (536) Google Scholar) (Figure S1C; Table S1). Similar results were obtained when THZ1 was tested against a larger panel of MYCN-amplified NB cell lines with varying levels of MYCN expression (Figures 1A and S1D). Importantly, NB cells without MYCN amplification were far less sensitive to THZ1, with IC50 values averaging ten times higher than those of MYCN-amplified cells (Figure 1A; Table S1). Notably, the NBL-S cell line, which expresses high levels of MYCN without genomic amplification (Cohn et al., 1990Cohn S.L. Salwen H. Quasney M.W. Ikegaki N. Cowan J.M. Herst C.V. Kennett R.H. Rosen S.T. DiGiuseppe J.A. Brodeur G.M. Prolonged N-myc protein half-life in a neuroblastoma cell line lacking N-myc amplification.Oncogene. 1990; 5: 1821-1827PubMed Google Scholar) (Figure S1D), was quite sensitive to THZ1, while two nontransformed lines (B6-MEFs and NIH 3T3) were relatively insensitive (Figure 1A). MYCN-amplified cells also showed enhanced sensitivity to THZ1R, a reversible analog of THZ1 that lacks the acrylamide moiety required for covalent bond formation, although it was not as potent as the covalent inhibitor (Figure 1B). The strong selectivity of THZ1 for MYCN-amplified cells was not restricted to NB, but extended to H262-BT111, a human primitive neuroectodermal tumor cell line expressing amplified MYCN (K. Ligon, personal communication) and Raji and Daudi lymphoma cells, both characterized by MYC overexpression due to chromosomal translocation (Nishikura et al., 1985Nishikura K. Erikson J. ar-Rushdi A. Huebner K. Croce C.M. The translocated c-myc oncogene of Raji Burkitt lymphoma cells is not expressed in human lymphoblastoid cells.Proc. Natl. Acad. Sci. USA. 1985; 82: 2900-2904Crossref PubMed Scopus (29) Google Scholar, Veronese et al., 1995Veronese M.L. Ohta M. Finan J. Nowell P.C. Croce C.M. Detection of myc translocations in lymphoma cells by fluorescence in situ hybridization with yeast artificial chromosomes.Blood. 1995; 85: 2132-2138Crossref PubMed Google Scholar) (Figure S1E). To probe this preferential effect further, we tested the extent of target engagement in NB cells, using a biotinylated derivative of THZ1 (bio-THZ1) with or without THZ1 pretreatment. Bio-THZ1 consistently bound to CDK7 in both MYCN-amplified and nonamplified untreated cells (Figure S1F), but became less efficient after THZ1 treatment, suggesting that target recognition was not a major factor in the enhanced inhibitory effects of THZ1 in MYCN-amplified cells.Figure 1THZ1 Exhibits High Potency and Selectivity against MYCN-Amplified Tumor CellsShow full caption(A) Dose-response curves of MYCN-amplified and nonamplified human NB and murine fibroblast cells after treatment with increasing concentrations of THZ1 for 72 hr. Percent cell viability relative to that of DMSO-treated cells is shown here and in (B). Data represent mean ± SD of three replicates here and in (B).(B) Dose-response curves of NB cells treated as in (A) with the reversible CDK7 inhibitor THZ1R.(C) Cell-cycle analysis of MYCN-amplified versus nonamplified NB cells exposed to THZ1 (100 nM × 24 and 48 hr) by flow cytometry with propidium iodide (PI) staining. Results are representative of three replicates. The scale and axes are indicated in the lower left corner.(D) Apoptosis analysis in MYCN-amplified and nonamplified NB cells treated with THZ1 as in (C) by flow cytometry with Annexin V staining. Data represent mean ± SD of three replicates. ∗∗∗p < 0.0001, ∗∗p < 0.001 (Student’s t test).(E) Tumor volumes of MYCN-amplified human NB xenografts in NU/NU (Crl:NU-Foxn1nu) mice treated with THZ1 (10 mg/kg intravenously [i.v.] twice daily) (n = 14) or vehicle (n = 9) for 28 days. Mean ± SD values are presented. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05 (multiple t test, Holm-Sidak method).(F) Immunohistochemical (IHC) analysis of morphology (hematoxylin & eosin [H&E]), proliferation (Ki67) and apoptosis (cleaved caspase 3 [CC3]) in tumors harvested from animals treated with vehicle or THZ1 as in (E) for 12 days. Scale bar represents 25 μM.See also Figure S1 and Table S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Dose-response curves of MYCN-amplified and nonamplified human NB and murine fibroblast cells after treatment with increasing concentrations of THZ1 for 72 hr. Percent cell viability relative to that of DMSO-treated cells is shown here and in (B). Data represent mean ± SD of three replicates here and in (B). (B) Dose-response curves of NB cells treated as in (A) with the reversible CDK7 inhibitor THZ1R. (C) Cell-cycle analysis of MYCN-amplified versus nonamplified NB cells exposed to THZ1 (100 nM × 24 and 48 hr) by flow cytometry with propidium iodide (PI) staining. Results are representative of three replicates. The scale and axes are indicated in the lower left corner. (D) Apoptosis analysis in MYCN-amplified and nonamplified NB cells treated with THZ1 as in (C) by flow cytometry with Annexin V staining. Data represent mean ± SD of three replicates. ∗∗∗p < 0.0001, ∗∗p < 0.001 (Student’s t test). (E) Tumor volumes of MYCN-amplified human NB xenografts in NU/NU (Crl:NU-Foxn1nu) mice treated with THZ1 (10 mg/kg intravenously [i.v.] twice daily) (n = 14) or vehicle (n = 9) for 28 days. Mean ± SD values are presented. ∗∗∗p < 0.001; ∗∗p < 0.01; ∗p < 0.05 (multiple t test, Holm-Sidak method). (F) Immunohistochemical (IHC) analysis of morphology (hematoxylin & eosin [H&E]), proliferation (Ki67) and apoptosis (cleaved caspase 3 [CC3]) in tumors harvested from animals treated with vehicle or THZ1 as in (E) for 12 days. Scale bar represents 25 μM. See also Figure S1 and Table S1. Next, we studied the growth inhibitory effects of THZ1. MYCN-amplified cells treated with THZ1 underwent cell-cycle arrest in G2/M at 24 hr, an effect that was not observed in MYCN-nonamplified NB cells, even after 48 hr (Figure 1C). Moreover, THZ1 led to a profound induction of apoptosis in high MYCN-expressing cells, but not in cells expressing nonamplified MYCN (Figures 1D and S1G). Together, these data indicate that THZ1 induces selective cytotoxicity not only in NB cells with MYCN amplification, but also in other cancers overexpressing either the MYCN or MYC oncogene. Given the relative lack of target specificity of past CDK inhibitors, leading to adverse effects in normal cells (Lapenna and Giordano, 2009Lapenna S. Giordano A. Cell cycle kinases as therapeutic targets for cancer.Nat. Rev. Drug Discov. 2009; 8: 547-566Crossref PubMed Scopus (742) Google Scholar), we assessed the tolerability of THZ1 in non-tumor-bearing mice (n = 6) treated with 10 mg/kg intravenously twice daily. No systemic toxicity was observed even after 4 weeks of continuous administration (data not shown). We next tested the therapeutic effects of THZ1 in xenograft models of MYCN-amplified human NB derived from subcutaneous flank injection of Kelly cells. When tumors reached an optimal size (mean volume, ∼150 mm3; range, 75–235 mm3), the animals were divided into two groups and treated with vehicle (n = 9) or THZ1 as above (n = 14). Treatment was continued for a mean of 20 days (range, 15–24 days) in the control group and 24 days (range, 20–28 days) in the THZ1 group. Mice receiving THZ1 had a statistically significant reduction in tumor growth, again without toxicity (Figure 1E). Two animals remained free of tumor recurrence at 35 and 128 days posttreatment. Tumors from vehicle-treated mice displayed histological features of human NB with poorly differentiated, small round blue cells displaying high mitotic activity (Figure 1F). By contrast, the vast majority of tumor cells in the THZ1-treated animals demonstrated necrosis, reduced proliferative activity and increased apoptosis. To ensure target engagement in the tumor cells, we used bio-THZ1 to pull down CDK7 in cell lysates from both vehicle- and THZ1-treated animals, noting decreased binding with bio-THZ1 in the latter (Figure S1H). We also confirmed that the lack of toxicity in the animal models did not reflect THZ1 selectivity for human CDK7, as bio-THZ1 formed a complex with murine CDK7 in cell lysates from NIH 3T3 cells treated with THZ1 (Figure S1H). These results demonstrate the feasibility of specifically targeting CDK7 in tumor cells driven by deregulated MYC or MYCN. As a transcriptional kinase, CDK7 exerts its effects through regulation of RNA Pol II-mediated transcriptional initiation and pause establishment, while also affecting elongation through its CAK activity on other transcriptional CDKs (Glover-Cutter et al., 2009Glover-Cutter K. Larochelle S. Erickson B. Zhang C. Shokat K. Fisher R.P. Bentley D.L. TFIIH-associated Cdk7 kinase functions in phosphorylation of C-terminal domain Ser7 residues, promoter-proximal pausing, and termination by RNA polymerase II.Mol. Cell. Biol. 2009; 29: 5455-5464Crossref PubMed Scopus (224) Google Scholar, Larochelle et al., 2012Larochelle S. Amat R. Glover-Cutter K. Sansó M. Zhang C. Allen J.J. Shokat K.M. Bentley D.L. Fisher R.P. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.Nat. Struct. Mol. Biol. 2012; 19: 1108-1115Crossref PubMed Scopus (259) Google Scholar, Palancade and Bensaude, 2003Palancade B. Bensaude O. Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation.FEBS. 2003; 270: 3859-3870Google Scholar). We observed a dose-dependent decrease in the initiation-associated serine 5 (S5) and serine 7 (S7) and the elongation-associated serine 2 (S2) Pol II phosphorylation in MYCN-amplified but not nonamplified cells treated with THZ1 (Figures 2A and S2A). Pol II CTD phosphorylation was also downregulated in tumor cells from animals treated with THZ1 (Figure 2B). Downregulation of CDK7 phosphorylation was seen in MYCN-amplified cells (Figure S2B), consistent with the finding that CDK7 is regulated by phosphorylation within its own activation (T) loop (Larochelle et al., 2012Larochelle S. Amat R. Glover-Cutter K. Sansó M. Zhang C. Allen J.J. Shokat K.M. Bentley D.L. Fisher R.P. Cyclin-dependent kinase control of the initiation-to-elongation switch of RNA polymerase II.Nat. Struct. Mol. Biol. 2012; 19: 1108-1115Crossref PubMed Scopus (259) Google Scholar). Phosphorylation of CDK9 was also decreased in MYCN-amplified cells (Figure S2C), reinforcing the effect of THZ1 on transcription elongation. Total protein levels of CDK9 were also decreased in these cells but not nonamplified cells, suggesting that THZ1-induced CDK7 inhibition might also target the transcription of CDK9 (Figure S2C).Figure S2THZ1 Affects General Transcription and Cell-Cycle Regulation in Cells Expressing Deregulated MYC, Related to Figure 2Show full caption(A) Western blot analysis of RNA Pol II CTD phosphorylation in MYCN-amplified (IMR-32) and nonamplified (SK-N-FI) cells treated with DMSO or THZ1 (50 or 100 nM) for the indicated times. β-tubulin was used as a loading control.(B) Western blot analysis of pCDK7 and CDK7 expression in MYCN-amplified (Kelly) and nonamplified (SH-SY5Y) NB cells treated with DMSO or THZ1 at the indicated concentrations and times.(C) Western blot analysis of total and phosphorylated CDK9 expression in MYCN-amplified (Kelly, IMR-32) and nonamplified (SH-SY5Y, SK-N-F1) cells treated with THZ1 at the indicated concentrations and times.(D) Western blot analysis of RNA Pol II CTD phosphorylation and MCL1 expression in Raji, Daudi and H262 BT111 cells following treatment with DMSO or THZ1 at the indicated doses for 6 hr.(E) Western blot analysis of the indicated proteins in MYCN-amplified NB cells overexpressing either wild-type CDK7 (CDK7WT) or the CDK7C312S mutation and treated with DMSO or 100 nM THZ1 for 6 hr.(F) Western blot analysis of phosphorylated and total CDK1 and CDK2 in Kelly and SH-SY5Y cells treated with THZ1 at the indicated doses for 6 hr.(G) Dose-response curves of MYCN-amplified (Kelly, IMR-32) and nonamplified (SH-SY5Y, SK-N-SH) human NB cells after treatment with increasing concentrations of triptolide (left) for 48 hr. and purvanolol (right) for 72 hr. Percent cell viability relative to that of DMSO-treated cells is shown. The results are reported as mean ± SD of 3 replicates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Western blot analysis of RNA Pol II CTD phosphorylation in MYCN-amplified (IMR-32) and nonamplified (SK-N-FI) cells treated with DMSO or THZ1 (50 or 100 nM) for the indicated times. β-tubulin was used as a loading control. (B) Western blot analysis of pCDK7 and CDK7 expression in MYCN-amplified (Kelly) and nonamplified (SH-SY5Y) NB cells treated with DMSO or THZ1 at the indicated concentrations and times. (C) Western blot analysis of total and phosphorylated CDK9 expression in MYCN-amplified (Kelly, IMR-32) and nonamplified (SH-SY5Y, SK-N-F1) cells treated with THZ1 at the indicated concentrations and times. (D) Western blot analysis of RNA Pol II CTD phosphorylation and MCL1 expression in Raji, Daudi and H262 BT111 cells following treatment with DMSO or THZ1 at the indicated doses for 6 hr. (E) Western blot analysis of the indicated proteins in MYCN-amplified NB cells overexpressing either wild-type CDK7 (CDK7WT) or the CDK7C312S mutation and treated with DMSO or 100 nM THZ1 for 6 hr. (F) Western blot analysis of phosphorylated and total CDK1 and CDK2 in Kelly and SH-SY5Y cells treated with THZ1 at the indicated doses for 6 hr. (G) Dose-response curves of MYCN-amplified (Kelly, IMR-32) and nonamplified (SH-SY5Y, SK-N-SH) human NB cells after treatment with increasing concentrations of triptolide (left) for 48 hr. and purvanolol (right) for 72 hr. Percent cell viability relative to that of DMSO-treated cells is shown. The results are reported as mean ± SD of 3 replicates. The decrease in Pol II phosphorylation after THZ1 treatment in MYCN-amplified cells coincided with the loss of the short-lived antiapoptotic protein MCL1 (Figure 2C). Similar effects on Pol II CTD phosphorylation and MCL1 levels were observed in Raji, Daudi, and H262-BTIII cells, all of which depend on MYC or MYCN overexpression and are sensitive to THZ1 (Figure S2D). Importantly, the effects of THZ1, including Pol II CTD phosphorylation and the induction of apoptosis, could be rescued by a mutation in the covalent binding site of CDK7 (CDK7C312S) (Figure S2E), indicating on-targe" @default.
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• W2170846478 date "2014-11-01" @default.
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• W2170846478 title "CDK7 Inhibition Suppresses Super-Enhancer-Linked Oncogenic Transcription in MYCN-Driven Cancer" @default.
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• W2170846478 doi "https://doi.org/10.1016/j.cell.2014.10.024" @default.
• W2170846478 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4243043" @default.
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• W2170846478 hasPublicationYear "2014" @default.
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