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Triple negative breast cancer (TNBC) is highly proliferative and genomically unstable, making these tumors particularly sensitive to anti-proliferative chemotherapies. For nearly 20 years, inhibitors of cyclin/cyclin dependent kinases (CDK) that target proliferation have been investigated for the treatment of a myriad of neoplasms with limited success due to dose limiting toxicities as a result of generally targeting CDKs. Agents that can selectively target proliferation and mitosis within cancer cells without inducing systemic toxicity should greatly improve patient outcomes from TNBC. Recently, selective CDK7 inhibitors have entered clinical trials for solid tumors promising to diminish toxicity by primarily inhibiting CDK7. Due to resistance mechanisms that will inevitably manifest during treatment, understanding the pathways controlled by CDK7 inhibition is imperative. CDK7 forms a complex with MAT1 and cyclin H to phosphorylate serine 2 and 7 on the C terminal domain of RNA Polymerase II and promote pause release during transcription of TNBC driver genes. With selective covalent CDK7 inhibitors such as THZ1, RNA Polymerase II remains un-phosphorylated reducing the expression of genes necessary for mitosis, proliferation and viability. While this has been proposed to be due to inhibition of super-enhancer function, we show here that inhibition of CDK7 increases genomic instability of TNBC cell lines by promoting multi-nucleation and micro-nucleation. This is associated with an arrest at G2/M as well as the suppression of mitosis-related gene expression. Together, these data reveal that the mechanism of action of CDK7 inhibitors is similar to conventional anti-mitotics. Because resistance mechanisms frequently occur in cancer settings, we also generated two models of resistance to CDK7 inhibitors using the TNBC models, MDA-MB-231 and MDA-MB-468. We show the transporter ABCG2 can be up-regulated with the development of resistance and that inhibition of this transporter restores sensitivity to CDK7 inhibition. These data suggest that blockade of multi-drug resistance proteins such as ABCG2, may be useful for ensuring responsiveness to CDK7 inhibitors. Further analysis of gene expression changes occurring in response to such inhibitors should reveal additional key target genes that define therapeutic response in this disease. Understanding the full effects of CDK7 inhibition and resistance mechanisms may lead to a selective approach for targeting proliferation of TNBC and improving patient outcomes from this disease. Citation Format: Bryan M Webb, Melyssa Shively, Katrina Piemonte, Jennifer M Sahni, Ruth A Keri. Cyclin dependent kinase 7 ( CDK7) inhibition promotes genomic instability and mitotic catastrophe in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-04-01.

Cyclin-dependent kinase 7 (CDK7) a member of the cyclin-dependent kinase family, forms a functional CDK-activating kinase (CAK) with cyclin H and MAT1, which regulates the cell cycle by phosphorylating other CDKs. CDK7 is also a component of transcription factor II H (TFIIH) that regulates transcriptional initiation and elongation through the phosphorylation of the RNA polymerase II C-terminal domain (CTD). Inhibition of CDK7 has been reported as a potential cancer therapeutic by addressing aberrant cell cycle regulation and transcription, as these abnormalities are considered main factors for tumor progression in specific types of cancer. Q901 is a novel highly selective CDK7 inhibitor in IND enabling development stage. Q901 shows extreme selectivity by singular inhibition of CDK7 among tested kinome panel. Q901 triggers G1 cell cycle arrest in specific cancer models in vitro and in vivo. Good correlation has been demonstrated between Q901 tumor growth inhibition and target engagement of a pharmacodynamics marker in treated models, suggesting CDK7 is a main driver of tumor progression in the selected models. The Q901 data supports the continued preclinical evaluation and potential of the compound as investigational treatment for patients with multiple solid tumors. Citation Format: Donghoon Yu, Yeejin Jeon, Dongsik Park, Mooyoung Seo, Wongyun Ahn, Jaeseung Kim, Choa Park, Euna Jeong, Sukjoon Yoon, Kiyean Nam. Development of highly selective CDK7 inhibitor Q901 for solid tumors [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 4855.

CDK7 is a member of the cyclin-dependent kinase family, which forms the CDK activating kinase (CAK) complex with Cyclin H and RING finger protein Mat1 to control cell cycle progression and transcription by phosphorylating other CDKs and RNA polymerase II. Upregulation of CDK7 frequently occurs in human gastric cancer (GC). In this study, we demonstrated that inhibition of CDK7 was able to induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the increasing intracellular reactive oxidative species (ROS) levels. Pretreatment with ROS scavenger N-acety-L-cysteine partly reversed GC cell apoptosis induced by CDK7 inhibition. In the nude mice, inhibition of CDK7 also suppressed the growth of xenograft tumors of GC. Overall, our data showed that inhibition of CDK7 in GC presented outstanding anticancer effect in vitro and in vivo, suggesting that CDK7 is a potential therapeutic target for GC patients. Citation Format: Jia-Rong Huang, Wen-Ji Zhang, Qi-Wei Jiang, Yang Yang, Meng-Ning Wei, Zhi Shi. Targeting cyclin-dependent kinase 7 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4912.

Biliary tract cancers, which encompass tumors of the gallbladder, extrahepatic ducts, and ampulla of Vater, are relatively rare tumors with a high fatality rate. Other than a close link with gallstones, the etiology of biliary tract cancers is poorly understood. We conducted a population-based case-control study in Shanghai, China, to examine whether genetic variants in several DNA repair genes are associated with biliary tract cancers or biliary stones. Genomic DNA from 410 patients with biliary tract cancers (236 gallbladder, 127 bile duct, and 47 ampulla of Vater), 891 patients with biliary stones, and 786 healthy subjects randomly selected from the Shanghai population were genotyped for putative functional single nucleotide polymorphisms in four DNA repair genes (MGMT, RAD23B, CCNH, and XRCC3). Of the five single nucleotide polymorphisms examined, only one (MGMT EX5-25C>T, rs12917) was associated with biliary tract cancer. Independent of gallstones, subjects carrying the CT genotype of the MGMT EX5-25C>T marker had a significantly reduced risk of gallbladder cancer [odds ratio (OR), 0.63; 95% confidence interval (95% CI), 0.41-0.97; P = 0.02] and nonsignificant reduced risks of bile duct (OR, 0.61; 95% CI, 0.35-1.06) and ampulla of Vater (OR, 0.85; 95% CI, 0.39-1.87) cancers. However, this marker was not associated with biliary stones, and the other markers examined were not significantly associated with either biliary tract cancers or stones. Findings from this population-based study in Shanghai suggest that MGMT gene variants may alter susceptibility to biliary tract cancer, particularly gallbladder cancer. Confirmation in future studies, however, is required. (Cancer Epidemiol Biomarkers Prev 2008;17(8):2123–7)

Despite the potential importance of the cell cycle and apoptosis pathways in brain tumor etiology, little has been published regarding brain tumor risk associated with common gene variants in these pathways. Using data from a hospital-based case-control study conducted by the National Cancer Institute between 1994 and 1998, we evaluated risk of glioma (n = 388), meningioma (n = 162), and acoustic neuroma (n = 73) with respect to 12 single nucleotide polymorphisms from 10 genes involved in apoptosis and cell cycle control: CASP8, CCND1, CCNH, CDKN1A, CDKN2A, CHEK1, CHEK2, MDM2, PTEN, and TP53. We observed significantly decreased risk of meningioma with the CASP8 Ex14-271A>T variant [odds ratio (OR)AT, 0.8; 95% confidence interval (95% CI), 0.5-1.2; ORAA, 0.5; 95% CI, 0.3-0.9; Ptrend = 0.03] and increased risk of meningioma with the CASP8 Ex13+51G>C variant (ORGC, 1.4; 95% CI, 0.9-2.1; ORCC, 3.6; 95% CI, 1.0-13.1; Ptrend = 0.04). The CT haplotype of the two CASP8 polymorphisms was associated with significantly increased risk of meningioma (OR, 1.7; 95% CI, 1.1-2.6), but was not associated with risk of glioma or acoustic neuroma. The CCND1 Ex4-1G>A variant was associated with increased risk for glioma, and the Ex8+49T>C variant of CCNH was associated with increased risk of glioma and acoustic neuroma. The MDM2 Ex12+162A>G variant was associated with significantly reduced risk of glioma. Our results suggest that common variants in the CASP8, CCND1, CCNH, and MDM2 genes may influence brain tumor risk. Future research in this area should include more detailed coverage of genes in the apoptosis/cell cycle control pathways. (Cancer Epidemiol Biomarkers Prev 2007;16(8):1655–61)

Hepatocellular carcinoma (HCC) remains a significant unmet medical need with few therapeutic options available. Micro RNA 21 (miR-21) has been shown to be upregulated in HCC, however, contribution of this onco-miR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miR-21) and used them to interrogate dependency on miR-21 in a panel of 20 commercially available HCC cell lines in vitro. Upon lipid-mediated transfection, anti-miR-21, but not its mismatched (MM) control, caused significant de-repression of known direct targets of miR-21 (ANKRD46, DDAH1, RECK1) and inhibited survival of a large subset of HCC cell lines. Treatment of these sensitive HCC cell lines with anti-miR-21 resulted in dose- and time-dependent induction of caspase 3/7 activity. In contrast, non-responder HCC cell lines failed to significantly upregulate caspase activity and maintained viability in the presence of anti-miR compound. Further analysis of responder cell lines revealed robust induction of cell death, inhibition of cell migration and suppression of clonogenic growth upon treatment with miR-21 inhibitor. To better understand the consequences of miR-21 suppression in HCC, we carried out global gene expression profiling of anti-miR-21 treated sensitive liver cancer cells. Striking enrichment in miR-21 targets was noted among upregulated transcripts. Gene ontology analysis identified key cellular processes affected by miR-21 inhibition, including deregulation of metabolic pathways. In addition to the induction of direct miR-21 targets, cyclin H was found to be significantly downregulated upon miR-21 inhibition in the majority of responder cell lines. We hypothesize that inhibition of cyclin H expression, while an indirect effect of miR-21 suppression, could contribute to the activity of anti-miR-21 compounds. In summary, our data suggest that inhibition of miR-21 merits further investigation in the treatment of hepatocellular carcinoma. Citation Format: Sonya Zabludoff, Timothy Wagenaar, Francisco Adrian, Charles Allerson, Heike Arlt, Raffaele Baffa, Bal Bhat, Hui Cao, Scott Davis, Carlos Garcia-Echeverria, Kathrin Heermeier, Shih-Min Huang, Lan Jiang, Eric Marcusson, Christiane Metz-Weidmann, Adam Pavlicek, Jack Pollard, Jennifer Rocnik, Sabine Scheidler, Chaomei Shi, Fangxian Sun, Tatiana Tolstykh, Qunyan Yu, Gang Zheng, Dmitri Wiederschain. Targeting miR-21 in hepatocellular carcinoma. [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 4787. doi:10.1158/1538-7445.AM2014-4787

The cell cycle progression is controlled by cyclin-dependent kinases (CDKs). CDK-Activating Kinase (CAK) is an enzyme complex that is capable of phosphorylating (activating) all CDKs and is essential for G1 and G2 CDK activities. CAK is composed of CDK7, cyclin H, and Mat 1. CDK (CDK1 and CDK2) activity is also controlled by two inhibitory phosphorylation sites (tyrosine 15 and threonine 14) on the CDK. Dephosphorylation of these two sites by cdc25c activates CDK activity. We have previously reported that TGFβ inhibits growth of human myeloid leukemia cells via downregulation of multiple CDKs and cyclins. In this study, we investigated the effect of TGFβ on activity and expression of CAK and cdc25, the two upstream molecules of cell cycle regulatory molecules in human myeloid leukemia cells. TGF-β inhibited the proliferation of TF-1 and MV4-11 accompanied by an increase in p27 level and decrease in CDK1, CDK2, CDK4, cyclin A, cyclin D3, and cylcin B. However, TGF-β had no effect on the expression of CDK7, CDK 9, CDK11, cyclin H, Mat1, and their complexes (CDK7-cyclin H and CDK7-Mat1) for the time period tested up to 72 hours, detected by Western blot and immunoprecipitation, respectively. There were no detectable changes in the phosphorylation status of CDK7, CDK9, CDK11 in the cells treated with TGF-β as compared with the cells without TGFβ. In contrast, these cells showed marked decrease in the levels of MAT1 and CDK7 in response to retinoic acid and phorbol ester (PMA) stimulation. On the other hand, TGFβ significantly downregulated expression of cdc25c starting from 3h and with a maximum inhibition being observed at 72h. We also observed a transient dephosphorylation (activation) of cdc25c followed by significant decrease in the amount of dephophorylated 25c at 48 and 72h. Taken together, our data suggest that TGFβ-induced growth inhibition of human myeloid leukemia cells is CAK-independent but is linked to inhibition of early entry into mitosis of the cells by downregulating cdc25c-CDK1 pathway. Most likely, the downregulation of multiple CDKs and cyclins by TGFβ is regulated at translational or posttranslational but not transcriptional levels, because TGFβ had no any effect on the expression and phosphorylation of the CDKs that are involved in regulation of transcription factor TFII (CDK7 and CDK11) and TAK/P-TEFb (CDK9), respectively. (Supported by Faculty Incentive Grant and NIH-NIGMS MBRS RISE: R25 GM059244-13, Barry University). Citation Format: Alejandra Toro, Daria Vasilyeva, Talia Guardia, Tamara Guardia, Jazmine Duran, Xiaotang Hu. Transforming growth factor-beta (TGFβ)-mediated growth inhibition of human myeloid leukemia cells is CAK-independent but links to cdc25c-CDK1 pathway. [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 3470. doi:10.1158/1538-7445.AM2014-3470

Loss of SNF5, a core subunit of the SWI/SNF ATP-dependent chromatin-remodeling complex, has been linked to development of malignant rhabdoid tumors (MRT). MRTs, a deadly form of pediatric cancer, can occur in almost any soft tissue of the body, including the brain. We have previously shown that introduction of SNF5, a known tumor suppressor gene, into deficient cell lines results in a G1 growth arrest through p21(CIP1/WAF1) mRNA induction. However, additional gene expression changes that may contribute to MRT development or may be involved in SNF5 cell cycle arrest remain uncharacterized. To discover novel binding targets of SNF5, we used a Biosciences Human Cell Cycle RT2Profiler PCR expression array to identify genes whose expression significantly changed upon reexpression of SNF5-GFP in the MRT cell line A204.1. The genes identified include p16(INK4A), p21(CIP1/WAF1), CCNG2, CCNH, CDK8, and HERC5. p16(INK4A) and p21(CIP1/WAF1) have been previously validated as direct binding targets of SNF5. Cyclin G2 (CCNG2) is a noncanonical cyclin that appears to negatively control cell cycle progression. HERC5 is involved in interferon signaling and ISGylation of proteins. CDK8 is a member of the mediator complex that plays a role in transcription. Cyclin H (CCNH) has been linked to development of some neural cancers. We developed an adenoviral vector system containing a novel HA tagged SNF5 construct. We used RT-PCR to validate the gene expression changes seen in the array in A204.1 and other MRT cell lines using this novel construct. An empty adenoviral vector was used as a control. While all genes showed increases in A204.1 after SNF5 reexpression, CCNG2 and CCNH mRNA levels increased in only a few other MRT cell lines. CDK8 expression levels were unchanged or decreased in all other MRT cell lines examined. HERC5 mRNA expression was greatly increased in all MRT cell lines at significantly higher levels than any of the other genes identified in this study. Chromatin immunoprecipitation (ChIP) analysis of the HERC5 and CCNG2 promoter regions in A204.1 confirmed direct SNF5 binding. Maximal enrichment was observed upstream of the transcriptional start site of both promoters. RNA Polymerase II also increased across the promoter regions in a pattern similar to SNF5 binding. Western blotting was also performed, confirming the upregulation of HERC5. CCNG2 protein levels did not follow mRNA expression, possibly because of tight regulation due to involvement in the cell cycle. Overall, we have identified the HERC5 and CCNG2 promoters as direct binding targets of SNF5 that may play roles in the development of this cancer. Interestingly, previous studies have shown that interferon signaling is activated upon SNF5 introduction into MRTs, indicating the potential biological relevance of HERC5. Future studies are needed to fully characterize their relationship to MRT oncogenesis and the mechanism of SWI/SNF regulation at their proximal promoter region. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4028. doi:10.1158/1538-7445.AM2011-4028