Journal articles on the topic 'CDK17'
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Liu, Mingfa, Zhennan Xu, Zepeng Du, Bingli Wu, Tao Jin, Ke Xu, Liyan Xu, Enmin Li, and Haixiong Xu. "The Identification of Key Genes and Pathways in Glioma by Bioinformatics Analysis." Journal of Immunology Research 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/1278081.
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Glioma is the most common malignant tumor in the central nervous system. This study aims to explore the potential mechanism and identify gene signatures of glioma. The glioma gene expression profile GSE4290 was analyzed for differentially expressed genes (DEGs). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were applied for the enriched pathways. A protein-protein interaction (PPI) network was constructed to find the hub genes. Survival analysis was conducted to screen and validate critical genes. In this study, 775 downregulated DEGs were identified. GO analysis demonstrated that the DEGs were enriched in cellular protein modification, regulation of cell communication, and regulation of signaling. KEGG analysis indicated that the DEGs were enriched in the MAPK signaling pathway, endocytosis, oxytocin signaling, and calcium signaling. PPI network and module analysis found 12 hub genes, which were enriched in synaptic vesicle cycling rheumatoid arthritis and collecting duct acid secretion. The four key genes CDK17, GNA13, PHF21A, and MTHFD2 were identified in both generation (GSE4412) and validation (GSE4271) dataset, respectively. Regression analysis showed that CDK13, PHF21A, and MTHFD2 were independent predictors. The results suggested that CDK17, GNA13, PHF21A, and MTHFD2 might play important roles and potentially be valuable in the prognosis and treatment of glioma.
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Liang, Kaiwei, Xin Gao, Joshua M. Gilmore, Laurence Florens, Michael P. Washburn, Edwin Smith, and Ali Shilatifard. "Characterization of Human Cyclin-Dependent Kinase 12 (CDK12) and CDK13 Complexes in C-Terminal Domain Phosphorylation, Gene Transcription, and RNA Processing." Molecular and Cellular Biology 35, no. 6 (January 5, 2015): 928–38. http://dx.doi.org/10.1128/mcb.01426-14.
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Cyclin-dependent kinase 9 (CDK9) and CDK12 have each been demonstrated to phosphorylate the RNA polymerase II C-terminal domain (CTD) at serine 2 of the heptad repeat, both in vitro and in vivo . CDK9, as part of P-TEFb and the super elongation complex (SEC), is by far the best characterized of CDK9, CDK12, and CDK13. We employed both in vitro and in vivo assays to further investigate the molecular properties of CDK12 and its paralog CDK13. We isolated Flag-tagged CDK12 and CDK13 and found that they associate with numerous RNA processing factors. Although knockdown of CDK12, CDK13, or their cyclin partner CCNK did not affect the bulk CTD phosphorylation levels in HCT116 cells, transcriptome sequencing (RNA-seq) analysis revealed that CDK12 and CDK13 losses in HCT116 cells preferentially affect expression of DNA damage response and snoRNA genes, respectively. CDK12 and CDK13 depletion also leads to a loss of expression of RNA processing factors and to defects in RNA processing. These findings suggest that in addition to implementing CTD phosphorylation, CDK12 and CDK13 may affect RNA processing through direct physical interactions with RNA processing factors and by regulating their expression.
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Lier, S., I. Rein, S. Lund, A. Lång, E. Lång, N. Meyer, A. Dutta, et al. "P10.12.A CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival." Neuro-Oncology 24, Supplement_2 (September 1, 2022): ii51. http://dx.doi.org/10.1093/neuonc/noac174.177.
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Abstract Background Glioblastoma multiforme (GBM) is the most prevalent and aggressive malignant tumor of the central nervous system. With a median survival of only one year, GBM patients have a particularly poor prognosis, highlighting a clear need for novel therapeutic strategies to target this disease. Transcriptional cyclin-dependent kinases (CDK), which phosphorylate key residues of RNA polymerase II (RNAPII) C-terminal domain (CTD), play a major role in sustaining aberrant transcriptional programs that are key to development and maintenance of cancer cells. Material and Methods We used pharmacological inhibition and genetic ablation to study effects of CDK12/CDK13 depletion on the proliferatory and migratory capacity of GBM cells and mouse xenografts. SLAM-seq, CUT&RUN and cell cycle assays were used to study the mechanistic effects of CDK12/CDK13 depletion in GBM cells. Results CDK12/CDK13 depletion markedly reduced the proliferatory and migratory capacity of GBM cells, as well as in vivo growth. CDK12/CDK13 inhibition potentiated existing chemotherapeutic treatments. Mechanistically, inhibition of CDK12/CDK13 leads to a genome-wide abrogation of RNAPII CTD phosphorylation, which in turn disrupts transcription and cell cycle progression in GBM cells. Conclusion These results provide proof-of-concept for the potential of CDK12 and CDK13 as therapeutic targets for GBM.
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Kohoutek, Jiri, and Dalibor Blazek. "Cyclin K goes with Cdk12 and Cdk13." Cell Division 7, no. 1 (2012): 12. http://dx.doi.org/10.1186/1747-1028-7-12.
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Fan, Zheng, Jennifer R. Devlin, Simon J. Hogg, Maria A. Doyle, Paul F. Harrison, Izabela Todorovski, Leonie A. Cluse, et al. "CDK13 cooperates with CDK12 to control global RNA polymerase II processivity." Science Advances 6, no. 18 (April 29, 2020): eaaz5041. http://dx.doi.org/10.1126/sciadv.aaz5041.
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The RNA polymerase II (POLII)–driven transcription cycle is tightly regulated at distinct checkpoints by cyclin-dependent kinases (CDKs) and their cognate cyclins. The molecular events underpinning transcriptional elongation, processivity, and the CDK-cyclin pair(s) involved remain poorly understood. Using CRISPR-Cas9 homology-directed repair, we generated analog-sensitive kinase variants of CDK12 and CDK13 to probe their individual and shared biological and molecular roles. Single inhibition of CDK12 or CDK13 induced transcriptional responses associated with cellular growth signaling pathways and/or DNA damage, with minimal effects on cell viability. In contrast, dual kinase inhibition potently induced cell death, which was associated with extensive genome-wide transcriptional changes including widespread use of alternative 3′ polyadenylation sites. At the molecular level, dual kinase inhibition resulted in the loss of POLII CTD phosphorylation and greatly reduced POLII elongation rates and processivity. These data define substantial redundancy between CDK12 and CDK13 and identify both as fundamental regulators of global POLII processivity and transcription elongation.
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Shah, Muzna, Muhammad Fazal Hussain Qureshi, Danish Mohammad, Mahira Lakhani, Tabinda Urooj, and Shamim Mushtaq. "CDKs family -a glimpse into the past and present: from cell cycle control to current biological functions." Asian Pacific Journal of Cancer Biology 5, no. 1 (February 25, 2020): 1–9. http://dx.doi.org/10.31557/apjcb.2020.5.1.1-9.
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Cyclin-dependent kinases (CDKs) are the catalytic subunits or protein kinases characterized by separate subunit “cyclin” that are essential for their enzymatic activity. CDKs play important roles in the control of cell cycle progression, cell division, neuronal function, epigenetic regulation, metabolism, stem cell renewal and transcription. However, they can accomplish some of these tasks independently, without binding with cyclin protein or kinase activity. Thus, so far, twenty different CDKs and cyclins have been reported in mammalian cells. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). In this review, we summarizes that how CDKs are traditionally involve their latest revelations, their functional diversity beyond cell cycle regulation and their impact on development of disease in mammals.
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Zhang, Bo, Xuelin Zhong, Moira Sauane, Yihong Zhao, and Zhi-Liang Zheng. "Modulation of the Pol II CTD Phosphorylation Code by Rac1 and Cdc42 Small GTPases in Cultured Human Cancer Cells and Its Implication for Developing a Synthetic-Lethal Cancer Therapy." Cells 9, no. 3 (March 4, 2020): 621. http://dx.doi.org/10.3390/cells9030621.
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Rho GTPases, including Rho, Cdc42, Rac and ROP subfamilies, are key signaling molecules in RNA polymerase II (Pol II) transcriptional control. Our prior work has shown that plant ROP and yeast Cdc42 GTPases similarly modulate Ser2 and Ser5 phosphorylation status of the C-terminal domain (CTD) of the Pol II largest subunit by regulating CTD phosphatase degradation. Here, we present genetic and pharmacological evidence showing that Cdc42 and Rac1 GTPase signaling modulates a similar CTD Ser2 and Ser5 phosphorylation code in cultured human cancer cells. While siRNA knockdown of Cdc42 and Rac1, respectively, in HeLa cells increased the level of CTD Ser phosphatases RPAP2 and FCP1, they both decreased the level of CTD kinases CDK7 and CDK13. In addition, the protein degradation inhibitor MG132 reversed the effect of THZ1, a CDK7 inhibitor which could decrease the cell number and amount of CDK7 and CDK13, accompanied by a reduction in the level of CTD Ser2 and Ser5 phosphorylation and DOCK4 and DOCK9 (the activators for Rac1 and Cdc42, respectively). Conversely, treatments of Torin1 or serum deprivation, both of which promote protein degradation, could enhance the effect of THZ1, indicating the involvement of protein degradation in controlling CDK7 and CDK13. Our results support an evolutionarily conserved signaling shortcut model linking Rho GTPases to Pol II transcription across three kingdoms, Fungi, Plantae and Animalia, and could lead to the development of a potential synthetic-lethal strategy in controlling cancer cell proliferation or death.
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Guiro, Joana, Mathias Fagbemi, Michael Tellier, Justyna Zaborowska, Stephanie Barker, Marjorie Fournier, and Shona Murphy. "CAPTURE of the Human U2 snRNA Genes Expands the Repertoire of Associated Factors." Biomolecules 12, no. 5 (May 14, 2022): 704. http://dx.doi.org/10.3390/biom12050704.
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In order to identify factors involved in transcription of human snRNA genes and 3′ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with these human snRNA genes including RNA polymerase II (pol II), Cyclin-Dependent Kinase 7 (CDK7), Negative Elongation Factor (NELF), Suppressor of Ty 5 (SPT5), Mediator 23 (MED23) and several subunits of the Integrator Complex. Suppressor of Ty 6 (SPT6); Cyclin K, the partner of Cyclin-Dependent Kinase 12 (CDK12) and Cyclin-Dependent Kinase 13 (CDK13); and SWI/SNF chromatin remodelling complex-associated SWI/SNF-related, Matrix-associated, Regulator of Chromatin (SMRC) factors were also enriched. Several polyadenylation factors, including Cleavage and Polyadenylation Specificity Factor 1 (CPSF1), Cleavage Stimulation Factors 1 and 2 (CSTF1,and CSTF2) were enriched by U2 gene CAPTURE. We have already shown by chromatin immunoprecipitation (ChIP) that CSTF2—and Pcf11 and Ssu72, which are also polyadenylation factors—are associated with the human U1 and U2 genes. ChIP-seq and ChIP-qPCR confirm the association of SPT6, Cyclin K, and CDK12 with the U2 genes. In addition, knockdown of SPT6 causes loss of subunit 3 of the Integrator Complex (INTS3) from the U2 genes, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes and helped to identify a functional role for SPT6.
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Quereda, Victor, Simon Bayle, Francesca Vena, Sylvia M. Frydman, Andrii Monastyrskyi, William R. Roush, and Derek R. Duckett. "Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer." Cancer Cell 36, no. 5 (November 2019): 545–58. http://dx.doi.org/10.1016/j.ccell.2019.09.004.
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Zhang, Tinghu, Nicholas Kwiatkowski, Calla M. Olson, Sarah E. Dixon-Clarke, Brian J. Abraham, Ann K. Greifenberg, Scott B. Ficarro, et al. "Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors." Nature Chemical Biology 12, no. 10 (August 29, 2016): 876–84. http://dx.doi.org/10.1038/nchembio.2166.
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Greenleaf, Arno L. "Human CDK12 and CDK13, multi-tasking CTD kinases for the new millenium." Transcription 10, no. 2 (October 22, 2018): 91–110. http://dx.doi.org/10.1080/21541264.2018.1535211.
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Varadarajan, Ramya, Joseph Ayeni, Zhigang Jin, Ellen Homola, and Shelagh D. Campbell. "Myt1 inhibition of Cyclin A/Cdk1 is essential for fusome integrity and premeiotic centriole engagement in Drosophila spermatocytes." Molecular Biology of the Cell 27, no. 13 (July 2016): 2051–63. http://dx.doi.org/10.1091/mbc.e16-02-0104.
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Regulation of cell cycle arrest in premeiotic G2 phase coordinates germ cell maturation and meiotic cell division with hormonal and developmental signals by mechanisms that control Cyclin B synthesis and inhibitory phosphorylation of the M-phase kinase, Cdk1. In this study, we investigated how inhibitory phosphorylation of Cdk1 by Myt1 kinase regulates premeiotic G2 phase of Drosophila male meiosis. Immature spermatocytes lacking Myt1 activity exhibit two distinct defects: disrupted intercellular bridges (fusomes) and premature centriole disengagement. As a result, the myt1 mutant spermatocytes enter meiosis with multipolar spindles. These myt1 defects can be suppressed by depletion of Cyclin A activity or ectopic expression of Wee1 (a partially redundant Cdk1 inhibitory kinase) and phenocopied by expression of a Cdk1F mutant defective for inhibitory phosphorylation. We therefore conclude that Myt1 inhibition of Cyclin A/Cdk1 is essential for normal fusome behavior and centriole engagement during premeiotic G2 arrest of Drosophila male meiosis. The novel meiotic functions we discovered for Myt1 kinase are spatially and temporally distinct from previously described functions of Myt1 as an inhibitor of Cyclin B/Cdk1 to regulate G2/MI timing.
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Praditsuktavorn, Pannee, Benet Pera, Nicholas Kwiatkowski, ShaoNing Yang, Tinghu Zhang, Nathanael Gray, and Leandro Cerchietti. "Transcription Regulation Targeting in Peripheral T Cell Lymphomas Induces Apoptosis and Sensitization to BCL2 Inhibitors." Blood 124, no. 21 (December 6, 2014): 810. http://dx.doi.org/10.1182/blood.v124.21.810.810.
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Abstract Peripheral T-cell lymphomas (PTCL) are clinically aggressive diseases with poor response to available (largely B-cell lymphoma–tailored) chemotherapy regimens and dismal survival. To identify active drugs for PTCL patients, we performed a cell-based progressive screen from a library of 105 anti-neoplastic drugs in clinical use. Primary screening was done in the PTCL-NOS cell line (OCI-Ly12), using three drug concentrations. We identified 3 active drug groups within the clinical-range limit: HDAC inhibitors (HDI) (romidepsin), proteasome inhibitors (bortezomib, carfilzomib) and transcription inhibitors (dactinomycin). Secondary screening was conducted for the active drug groups with 6 drug concentrations, in an extended panel of 9 TCL cell lines. We also expanded the drugs in each group by adding vorinostat, panobinostat and valproic acid for HDI and SNS032 (CDK9>2>>7 inhibitor) and THZ1 (CDK7>12 inhibitor) for transcription inhibitors. We demonstrated that the most active drugs (within serum achievable concentrations) were bortezomib, carfilzomib, romidepsin, dactinomycin and THZ1. Since proteasome inhibitors and romidepsin are being currently tested in PTCL clinical trials, we focused on the transcriptional inhibitors. Actinomycin inhibits the transcription initiation complex and prevents RNA elongation by RNA-POL2; given this broad mechanism of action, it is associated with serious side effects that limit its clinical use either alone or in combination. On the other hand, a recently discovered CDK7>12 inhibitor, THZ1, showed minimal side effects in vivo (Kwiatkowski N, et al. Nature 2014 Jun22), and high potency in our secondary screening yielding IC50s in TCL of ~200nM. We therefore used THZ1 to investigate the functional relevance of CDK7/12 targeting in PTCL. CDK7 is a critical component of both CDK-activating kinase (CAK) and transcription factor II human (TFIIH) complexes that phosphorylates cell cycle CDKs and C-terminal domain (CTD) of RNA-POL2, respectively. CDK7 inhibition decreases the phosphorylation of RNA-POL2 at Ser5 and Ser2, leading to transcriptional inhibition of susceptible loci. CDK12 in complex with CCNK displays a CTD kinase activity that is required for RNA splicing. To better understand the sensitivity of TCL to THZ1, we first determined the expression of all CAK components (CDK7, CCNH, and MAT1A) and CDK12/CCNK in 9 TCL cell lines that were found overexpressed compared to normal T cells from tonsils. Treatment of OCI-Ly12 and OCI-Ly13.2 (ALCL-ALK negative) cell lines with THZ1 for up to 48 h induced dose- and time-dependent decrease of phospho-POL2-ser5 and phospho-POL2-ser2, followed by PARP cleavage, caspase 7/3 activation and apoptosis. Genes with super-enhancers were found to be more susceptible to THZ1, and we also found these enhancers in genes associated with PTCL prognosis such as MCL1, JAK1 and MYC. Accordingly, THZ1 decreased mRNA and protein levels of MCL1, JAK1 and MYC as early as 3 hours after treatment. This was followed by decreasing levels of BCL2, BCL-XL, JUND and NFkB, and increased expression of pro-apoptotic proteins such as BAX at later time points. The decrease in JAK1 abundance led up to 70% reduction of phospho-STAT3 activity (as determined by immunoblotting and EMSA-like assays). Moreover, induction of STAT3 phosphorylation by IL-7/IL-15 treatment partially rescued the effect of THZ1, suggesting that JAK1 is a relevant target of CDK7 in PTCLs. The THZ1-dependent decrease in anti-apoptotic BCL2-family members prompted us to determine whether CDK7/12 inhibition can re-sensitize TCL to these drugs. We found either synergistic or re-sensitization effects on combination of THZ1 with BH3-family inhibitors, ABT-737 and obatoclax, in both OCI-LY12 and OCI-Ly13.2 cells. In sum, we identified a mechanism by which CDK7/12 inhibition, with the irreversible clinical candidate compound THZ1, simultaneously inhibits prominent PTCL survival pathways (JAK/STAT3, MYC and BCL2) causing apoptosis and re-sensitization to BCL2-family inhibitors. Disclosures Off Label Use: THZ1 (CDK7 and CDK12 inhibitor) for inducing apoptosis and sensitization to BCL2 inhibitors in peripheral T cell lymphomas.. Kwiatkowski:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Zhang:Syros Pharmaceuticals: One of the inventors Patents & Royalties. Gray:Syros Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
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Chen, Hong-Ru, Guan-Ting Lin, Chun-Kai Huang, and Ming-Ji Fann. "Cdk12 and Cdk13 regulate axonal elongation through a common signaling pathway that modulates Cdk5 expression." Experimental Neurology 261 (November 2014): 10–21. http://dx.doi.org/10.1016/j.expneurol.2014.06.024.
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Kapasi, Anokhi J., and Deborah H. Spector. "Inhibition of the Cyclin-Dependent Kinases at the Beginning of Human Cytomegalovirus Infection Specifically Alters the Levels and Localization of the RNA Polymerase II Carboxyl-Terminal Domain Kinases cdk9 and cdk7 at the Viral Transcriptosome." Journal of Virology 82, no. 1 (October 17, 2007): 394–407. http://dx.doi.org/10.1128/jvi.01681-07.
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ABSTRACT We previously reported that defined components of the host transcription machinery are recruited to human cytomegalovirus immediate-early (IE) transcription sites, including cdk9 and cdk7 (S. Tamrakar, A. J. Kapasi, and D. H. Spector, J. Virol. 79:15477-15493, 2005). In this report, we further document the complexity of this site, referred to as the transcriptosome, through identification of additional resident proteins, including viral UL69 and cellular cyclin T1, Brd4, histone deacetylase 1 (HDAC1), and HDAC2. To examine the role of cyclin-dependent kinases (cdks) in the establishment of this site, we used roscovitine, a specific inhibitor of cdk1, cdk2, cdk7, and cdk9, that alters processing of viral IE transcripts and inhibits expression of viral early genes. In the presence of roscovitine, IE2, cyclin T1, Brd4, HDAC1, and HDAC2 accumulate at the transcriptosome. However, accumulation of cdk9 and cdk7 was specifically inhibited. Roscovitine treatment also resulted in decreased levels of cdk9 and cdk7 RNA. There was a corresponding reduction in cdk9 protein but only a modest decrease in cdk7 protein. However, overexpression of cdk9 does not compensate for the effects of roscovitine on cdk9 localization or viral gene expression. Delaying the addition of roscovitine until 8 h postinfection prevented all of the observed effects of the cdk inhibitor. These data suggest that IE2 and multiple cellular factors needed for viral RNA synthesis accumulate within the first 8 h at the viral transcriptosome and that functional cdk activity is required for the specific recruitment of cdk7 and cdk9 during this time interval.
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hu, Shan, David Moebius, Wojciech Dworakowski, Elliott Cooper, Derek LaPlaca, Sydney Alnemy, Phone Perera, et al. "Abstract 5393: An oral and selective CDK12 inhibitor demonstrates robust anti-tumor activity." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5393. http://dx.doi.org/10.1158/1538-7445.am2022-5393.
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Abstract Background: CDK12 has emerged as an attractive cancer target due to its role in transcription and DNA damage repair regulation. In pre-clinical models, small molecule CDK12 inhibitors have demonstrated promising tumor killing effect, especially in combination with DNA damaging agents. Here we profiled new oral and selective CDK12 inhibitors. Material and methods: ADP-glo࣪ assays: inhibition of recombinant CDK12/CyclinK, CDK2/CyclinE, CDK7/CyclinH/MAT1 and CDK9/CyclinT2 was measured by assessing ADP converted from ATP via luminescent signal at Km and 20x Km ATP. Kinome screen: 1 µM of Compound A was tested in duplicate against 370 kinases at 10 µM ATP in the radiometric HotSpot™ assay. Cellular assays: p-Ser2, p-Ser5 and total RNA pol II signals were assessed 4hrs after compound treatment while ɣH2AX and BRCA1 signals were measured after 24-48hr compound treatment via Immunofluorescence (IF) assays. Cell proliferation was determined using cell titer glo after 48-72hrs compound treatment. Apoptosis was measured by annexin V/PI staining and flow cytometry analysis after 48-72hrs of treatment. Cell cycle profile was evaluated with Click-iT-EdU and FxCycle violet stain and flow cytometry analysis following 24-48hrs of treatment. Mouse xenograft: balb/c mice were implanted subcutaneously with H1048 or MDA-MB-468 cells and randomized for treatment with test drug or vehicle when tumors reached 150-200mm3. Mice were dosed BID through oral administration for 4 weeks. Combination effect of Compound A and lurbinectedin was tested in vivo. Lurbinectedin and Compound A were dosed QW and BID respectively for 28 days in H1048 CDX model. Results: A series of CDK12 inhibitors were designed and profiled in biochemical and cellular assays. A representative member of the class, Compound A, exhibited selectivity over CDK2, CDK7, and CDK9 of 46-, 27-, and 9-fold, respectively. Compound A inhibited proliferation in a panel of cell lines with EC50 in the low nanomolar range. Compound A treatment led to dose dependent apoptosis in multiple cancer cell lines and induced G2/M arrest in cancer cell lines. In vitro, combination treatment with Compound A and lurbinectedin lead to increased DNA damage accumulation, decreased homologous recombination repair, and overall enhanced antiproliferation. Dose dependent tumor growth inhibition in SCLC and TNBC CDX models was observed with Compound A treatment. Enhanced and durable antitumor effect was observed with lurbinectedin and Compound A combination compared to single agent treatment in vivo. Conclusions: We designed and profiled orally available, CDK12 selective inhibitors with potent activity as single agent in vitro and in vivo in multiple cancer models. Compound A demonstrated enhanced antitumor effect in vitro and in vivo when combined with DNA damaging agents. These data support the rationale for advancing one or more members of this class toward clinical development. Citation Format: Shan hu, David Moebius, Wojciech Dworakowski, Elliott Cooper, Derek LaPlaca, Sydney Alnemy, Phone Perera, Jason Marineau, Claudio Chuaqui, John P. Carulli, Eric Olson. An oral and selective CDK12 inhibitor demonstrates robust anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5393.
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Kolloch, Lina, Teresa Kreinest, Michael Meisterernst, and Andrea Oeckinghaus. "Control of Expression of Key Cell Cycle Enzymes Drives Cell Line-Specific Functions of CDK7 in Human PDAC Cells." International Journal of Molecular Sciences 23, no. 2 (January 12, 2022): 812. http://dx.doi.org/10.3390/ijms23020812.
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Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.
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Stern, Yaakov E., Pompom Ghosh, Hannah L. Walker-Mimms, John W. Mosior, Denis Imbody, Hitendra S. Solanki, Andrii Monastyrskyi, Derek R. Duckett, and Eric B. Haura. "Abstract B028: CDK12/13 inhibition antagonizes resistance to KRASG12C inhibitors." Cancer Research 82, no. 23_Supplement_2 (December 1, 2022): B028. http://dx.doi.org/10.1158/1538-7445.cancepi22-b028.
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Abstract Covalent inhibitors selectively targeting the KRASG12C mutation offer a promising new therapeutic opportunity for non-small cell lung cancer (NSCLC) patients, with partial or complete response observed in up to 40% of cancers harboring this lesion. As observed for other precision medicines targeting oncogenic drivers, resistance to these agents develops upon prolonged treatment. We have developed cell-based models of acquired resistance to the KRASG12C inhibitor sotorasib by serial passage of sotorasib-sensitive NSCLC cell lines in the presence of the drug. We have observed dynamic phosphorylation of the transcriptional elongation kinases CDK12 and CDK13 as well as hyperphosphorylation of a network of DNA damage response (DDR) proteins in sotorasib-resistant cell lines by phosphoproteomic profiling of resistant and sensitive cells. This is accompanied by elevated sensitivity to DDR pathway and CDK12/13 (e.g., SR-4835) inhibitors. Combined treatment of NSCLC cell lines with sotorasib and SR-4835 delays or prevents adaptation to sotorasib and the development of acquired resistance. Furthermore, gene expression profiling of SR-4835-treated cells reveals that CDK12/13 inhibition suppresses both DDR gene expression and metabolic pathways associated with resistance to sotorasib. This reflects synergy between SR-4835 and inhibitors targeting the DDR pathway in breast cancer cell lines and demonstrates that transcriptional elongation is a critical vulnerability for cells undergoing adaptation to KRASG12C inhibitors via DDR-dependent mechanisms. Citation Format: Yaakov E. Stern, Pompom Ghosh, Hannah L. Walker-Mimms, John W. Mosior, Denis Imbody, Hitendra S. Solanki, Andrii Monastyrskyi, Derek R. Duckett, Eric B. Haura. CDK12/13 inhibition antagonizes resistance to KRASG12C inhibitors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B028.
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Ogi, Sayaka, Yasuhiro Aga, Kazuhiro Onuma, Hidetoshi Sunamoto, Takashi Matsushita, Ayumi Ogawa, Tohru Hasegawa, et al. "Preclinical in vitro and in vivo evaluation of antitumor activity of UD-017, a novel selective and orally available CDK7 inhibitor, in colorectal cancer." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e14085-e14085. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e14085.
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e14085 Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for cancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA procession. We synthesized UD-017, a small molecule, highly selective CDK7 inhibitor with a novel chemotype. In this study, we characterize antitumor activity of UD-017 in vitro and in vivo, and try to elucidate underlying mechanisms by which CDK7 inhibition contributes to the antitumor efficacy in colorectal cancer cells. Methods: We examined CDK7 selectivity of UD-017 against the other CDKs and kinases. We evaluated the antitumor activity in a variety of human cancer cell lines including colorectal cancer cells. We investigated the mechanism of antitumor activity of UD-017 using a human colorectal cancer cell line, HCT-116. In vivo antitumor efficacy of UD-017 was assessed in HCT-116 xenograft models and patient derived xenograft (PDX) model. Results:UD-017 inhibited CDK7 enzyme (IC50 = 16 nM), which is at least 300-fold more selective against other CDKs. In a panel of 313 kinases assay, UD-017 inhibited CDK7 almost mono-specifically. UD-017 potently inhibited the growth of human cancer cells including the patient-derived colorectal cancer cells.In the mechanism study, UD-017 inhibited phosphorylation of CDK1, 2 and retinoblastoma, which was followed by cell-cycle arrest. Inhibition of both RNA polymerase II phosphorylation and c-Myc expression was observed followed by apoptosis induction. HCT-116 xenograft model and PDX model demonstrated the potent antitumor activity of UD-017 with dose-dependence by daily oral administration for 2 weeks. Furthermore, UD-017 showed the combination effect with 5-FU without further affecting the side effects of the chemotherapy. Conclusions:UD-017 is a potent and highly selective CDK7 inhibitor, and significantly inhibits the growth of human cancer cell lines with cell-cycle arrest and apoptosis induction. UD-017 showed potent and dose-dependent antitumor response in the HCT-116 xenograft model and PDX model. Further investigation towards clinical development is warranted.
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Du, Jianhai, Na Wei, Tongju Guan, Hao Xu, Jianzhong An, Kirkwood A. Pritchard, and Yang Shi. "Inhibition of CDKS by roscovitine suppressed LPS-induced ·NO production through inhibiting NFκB activation and BH4 biosynthesis in macrophages." American Journal of Physiology-Cell Physiology 297, no. 3 (September 2009): C742—C749. http://dx.doi.org/10.1152/ajpcell.00138.2009.
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In inflammatory diseases, tissue damage is critically associated with nitric oxide (·NO) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on ·NO production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of ·NO induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of IκB kinase β (IKKβ), IκB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH2-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1β, and IL-6 but not tumor necrosis factor (TNF)-α. Tetrahydrobiopterin (BH4), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH2). Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4-to-BH2 ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH4 biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced ·NO production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited ·NO production, iNOS, and COX-2 upregulation, activation of NFκB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced ·NO production in macrophages by suppressing nuclear factor-κB activation and BH4 biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.
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Yamakawa, Hiroko, Akio Mizutani, Yasuyoshi Arikawa, Shunsuke Ebara, Yoshihiko Satoh, and Daisuke Morishita. "Abstract 5485: Discovery and preclinical evaluation of a novel highly selective and potent CDK12 inhibitor." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5485. http://dx.doi.org/10.1158/1538-7445.am2022-5485.
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Abstract Recent research highlights that RNA processing is systematically altered in cancer, demonstrating the pivotal influence of RNA deregulation on tumorigenesis, growth and progression. Based on our expanding knowledge of RNA biology, RNA deregulation has drawn much attention from the perspective of cancer therapy. Indeed, small molecules that attack the RNA maturation processes and produce aberrant RNA are currently under development. The first step in gene expression is transcription, which involves copying a DNA sequence to make an RNA. Transcription is performed by enzymes called RNA polymerase (Pol II) that links nucleotides to form RNA strand. The C-terminal domain (CTD) of Pol II comprises heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 repeats and is dynamically post-translationally phosphorylated to regulate the distinct stages of transcription initiation, elongation and termination steps. Particularly, Ser2 and Ser5 phosphorylation have the closest association with the regulation of transcription. Therefore, we focus on the kinases that carry out this modification. Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. CDK12 regulates the elongation step of RNA transcription by phosphorylation of Ser2 on the CTD. CDK12 complexes with cyclin K to promote the elongation of transcripts, such as BRCA1 and BRCA2, involved in DNA damage responses. It is expected that the inhibition of CDK12 have a synergistic effect with PARP inhibitors and chemotherapeutic reagents. Here we present data on CRD-1835439, orally available, selective, and first in class CDK12 inhibitor with optimized drug-properties. Biochemically, CRD-1835439 is more than 300-fold selective over other CDK isoforms such as CDK7 and CDK9. Importantly, cell based assays showed CRD-1835439 has selective inhibitory activity for phosphorylation of Ser2 on the CTD but not for other Ser5 and Ser7 phosphorylation. Comprehensive analysis by PolyA-seq, Chip-seq and RNA-seq revealed that CRD-1835439 inhibits transcriptional elongation on DNA damage response genes including BRCA1 and BRCA2. The effects on DNA damage and repair were assessed by immunofluorescence staining for γH2AX and RAD51 proteins. In vivo, oral treatment with CRD-1835439 in cell line derived xenograft models, resulted in increase of DNA damage biomarkers, induction of apoptosis and tumor regressions in a dose-dependent fashion. Besides the efficacy as a single reagent, the efficacy was augmented when CRD-1835439 was combined with PARP inhibitors in in vitro and in vivo. In summary, the novel small-molecule CDK12 inhibitor CRD-1835439 demonstrated preclinical efficacy along with target engagement. Our results underscore the preclinical therapeutic potential of CRD-1835439 as a single-agent or in combination with PARP inhibitors for the treatment of intractable cancers. Citation Format: Hiroko Yamakawa, Akio Mizutani, Yasuyoshi Arikawa, Shunsuke Ebara, Yoshihiko Satoh, Daisuke Morishita. Discovery and preclinical evaluation of a novel highly selective and potent CDK12 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5485.
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Yao, Yao, Woojun D. Park, Eugenio Morelli, Mehmet Kemal Samur, Nicholas P. Kwiatkowski, Yan Xu, Chandraditya Chakraborty, et al. "Targeting MM at the Nexus between Cell Cycle and Transcriptional Regulation Via CDK7 Inhibition." Blood 136, Supplement 1 (November 5, 2020): 1–2. http://dx.doi.org/10.1182/blood-2020-142592.
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Deregulated transcription and cell cycle control are hallmarks of cancer that are especially frequent in multiple myeloma (MM). Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription; and targeting of cell cycle CDKs has been long pursued as an attractive therapeutic strategy. Among CDKs, CDK7 presents a unique therapeutic opportunity as it functions as a CDK activating kinase (CAK), licensing the activity of cell cycle CDKs, and also serves as a core component of the general transcription factor TFIIH. Here we elucidated the biological role of CDK7 and its transcriptional regulatory landscape in MM, using genetic as well chemical approaches, including tools for CDK7 rapid protein degradation (dTAG) and the selective covalent inhibitor YKL-5-124 that targets a cysteine residue (C312) located outside of the kinase domain. We have observed that CDK7 inhibition via YKL-5-124 robustly inhibited the phosphorylation of the CDK1, 2 and 4 activation loops in a representative panel of MM cell lines at concentrations as low as 50 nM. This reduction was not observed in MM cells expressing a resistant mutation in the reactive cysteine (C312S). Consistent with decrease of CAK activity, we observed G1 arrest and S phase loss after CDK7 inhibition, which was also associated with a rapid and transient loss of Ser2 and Ser5 phosphorylation of the RNA Pol2 C-terminal domain. To understand the effect of CDK7 inhibition on MM cell growth and viability, we evaluated activity of YKL-5-124 across a large panel of 25 MM cell lines and observed a significant inhibition of MM cell proliferation, with a significantly lower IC50 compared to PHA-activated normal donor peripheral blood mononuclear cells (PBMCs), suggesting a specific sensitivity of MM cells to CDK7 inhibition. Longer exposure to YKL-5-124 caused apoptotic cell death in MM cells; however treatment with an inactive analog or in cells expressing the C312S mutation failed to inhibit MM cell proliferation, confirming that the antiproliferative potency of YKL-5-124 resides in its unique characteristic to covalently bind to C312 domain. Importantly, CDK7 inhibition impaired primary MM cells proliferation alone and when cultured in the presence of BM microenvironment. Selective pharmacological degradation of endogenously tagged CDK7 confirmed impact of CDK7 inhibition on MM cell proliferation via inhibition of CDK7 transcriptional and cell cycle activities. To complement the pharmacological studies, we have established MM cells to express inducible CRISPR/Cas9 constructs encoding 4 independent small guide RNAs targeting CDK7, resulting in the reduction of the abundance of CDK7 protein by 20-60% which was sufficient to inhibit MM cell viability over time, phenocopying pharmacologic inhibition of CDK7. These results support the view that CDK7 is a pharmacologically relevant target for MM. Gene expression analysis after CDK7 inhibition in MM1S and H929 cells revealed that transcripts for only a subset of genes were substantially affected by treatment with low dose of YKL-5-124, showing a strong leading-edge enrichment for downregulation of E2F expression program, cell cycle, DNA damage, and MYC targets. We have indeed confirmed a potent reduction in phosphorylation of RB protein, with consequent decrease of E2F activity in MM cells confirmed using E2F-driven luciferase reporter. These data suggest significant role for CDK7 in the CDK-pRB-E2F pathway in MM, which was strengthened by the observation of a positive correlation between expression of CDK7 and expression of E2F target genes in primary MM cells (n=409). Finally, we have evaluated the in vivo effect of CDK7 inhibition in several murine models of human MM. In the localized subcutaneous model, and the disseminated MM model where treatment with YKL-5-124 decreased tumor burden and improved survival. The effect of CDK7 inhibition explored in an aggressive, genetically engineered model of Myc-dependent MM, revealed evidence of response by decline in measurement of monotypic serum immunoglobulins. In conclusion, our study demonstrates that CDK7 contributes to the 'transcriptional addiction' and the cell cycle deregulation frequently observed in MM and represents an attractive molecular vulnerability to be exploited therapeutically. Disclosures Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Oncopep and C4 Therapeutics.: Other: Scientific Founder of Oncopep and C4 Therapeutics.; Celgene: Membership on an entity's Board of Directors or advisory committees. Munshi:Takeda: Consultancy; Karyopharm: Consultancy; AbbVie: Consultancy; Amgen: Consultancy; Legend: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; C4: Current equity holder in private company; OncoPep: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Consultancy. Fulciniti:NIH: Research Funding.
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Li, Ji, Porter, Broude, Roninson, and Chen. "Characterizing CDK8/19 Inhibitors through a NFκB-Dependent Cell-Based Assay." Cells 8, no. 10 (October 6, 2019): 1208. http://dx.doi.org/10.3390/cells8101208.
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Cell-based assays for CDK8/19 inhibition are not easily defined, since there are no known cellular functions unique to these kinases. To solve this problem, we generated derivatives of 293 cells with CRISPR knockout of one or both of CDK8 and CDK19. Double knockout (dKO) of CDK8 and CDK19 together (but not individually) decreased the induction of transcription by NFκB (a CDK8/19-potentiated transcription factor) and abrogated the effect of CDK8/19 inhibitors on such induction. We generated wild type (WT) and dKO cell lines expressing luciferase from an NFκB-dependent promoter. Inhibitors selective for CDK8/19 over other CDKs decreased TNFα-induced luciferase expression in WT cells by ~80% with no effect on luciferase induction in dKO cells. In contrast, non-selective CDK inhibitors flavopiridol and dinaciclib and a CDK7/12/13 inhibitor THZ1 (but not CDK4/6 inhibitor palbociclib) suppressed luciferase induction in both WT and dKO cells, indicating a distinct role for other CDKs in the NFκB pathway. We used this assay to characterize a series of thienopyridines with in vitro bone anabolic activity, one of which was identified as a selective CDK8/19 inhibitor. Thienopyridines inhibited luciferase induction in the WT but not dKO cells and their IC50 values in the WT reporter assay showed near-perfect correlation (R2 = 0.98) with their reported activities in a bone anabolic activity assay, confirming that the latter function is mediated by CDK8/19 and validating our assay as a robust and quantitative method for CDK8/19 inhibition.
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Johannessen, Liv, Nan Ke, Priyanka Sawant, Wojciech Dworakowski, Anthony D'Ippolito, Shanhu Hu, Nisha Rajagopal, Matthew Eaton, and Graeme Hodgson. "Activity of SY-5609, an oral, noncovalent, potent, and selective CDK7 inhibitor, in preclinical models of colorectal cancer." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 3585. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.3585.
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3585 Background: Colorectal cancer (CRC) is driven by genetic alterations that result in constitutive activation of oncogenic transcription factors (eg β-catenin, MYC) and of mitogenic signaling and cell cycle progression (driven by oncogenic mutations in KRAS and BRAF). CDK7 is a key regulator of transcription, through phosphorylation of the CTD domain of RNA Polymerase II, and of cell cycle progression, through phosphorylation of the cell cycle kinases CDK1, 2, 4, and 6. This dual role of CDK7 suggests inhibitors of CDK7 may be effective in the treatment of CRC. SY-5609 is an oral, noncovalent, potent and highly selective CDK7 inhibitor in phase 1 clinical development for patients with advanced solid tumors including CRC (NCT04247126). Here we report on the activity of SY-5609 in patient-derived xenograft (PDX) models of CRC. Methods: SY-5609 was administered once daily (QD) by oral gavage for 21 days (end of treatment, EOT) to mice bearing PDX models of CRC. The relationship between SY-5609 dose, pharmacodynamic (PD) changes in xenograft tissue, tumor growth inhibition (TGI), and mouse body weight (BW) was evaluated across a range of doses. SY-5609 TGI activity was also evaluated at sub-maximum-tolerated-dose levels across a panel of 30 independent CRC models including BRAF-, KRAS-, and non-BRAF/KRAS-mutant (wild type) models (n = 10 per group). Results: SY-5609 induced dose-dependent TGI in BRAF-mutant CRC PDX tumors, with tumor regressions observed at well tolerated doses (no BW loss at EOT), and no tumor regrowth for 2+ weeks after treatment was discontinued. Dose-dependent TGI was associated with dose-dependent PD changes in PDX tumor tissue. Across 30 PDX models, SY-5609 at well-tolerated doses (average BW loss of 0% at EOT across all models) induced ≥50% TGI in 67% (20/30) of models. Deep responses (≥90% TGI or regressions) were observed in 23% (7/30) of models, with enrichment for deep responses in BRAF mutant models (50%, 5/10) relative to KRAS mutant (10%, 1/10), and wild type (10%, 1/10) models. Conclusions: Daily oral dosing of the CDK7 inhibitor SY-5609 induces robust TGI, including regressions, in CRC PDX models at well-tolerated doses. Dose-dependent TGI is associated with dose-dependent PD changes in CRC PDX tumor tissue. These results highlight the therapeutic potential of SY-5609 in CRC and support the evaluation of SY-5609 in CRC patients in early phase clinical trials. SY-5609 is in phase 1 clinical development for patients with advanced solid tumors including CRC (NCT04247126).
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Li, Tao-Sheng, Kimikazu Hamano, Masahiko Nishida, Masanori Hayashi, Hiroshi Ito, Akihito Mikamo, and Masunori Matsuzaki. "CD117+ stem cells play a key role in therapeutic angiogenesis induced by bone marrow cell implantation." American Journal of Physiology-Heart and Circulatory Physiology 285, no. 3 (September 2003): H931—H937. http://dx.doi.org/10.1152/ajpheart.01146.2002.
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Therapeutic angiogenesis can be induced by the implantation of bone marrow mononuclear cells. We investigated the roles of mature mononuclear cell and stem cell fractions in bone marrow in this treatment. Although CD34 is the most popular marker for stem cell selection for inducing therapeutic angiogenesis, we separated CD117-positive cells (CD117+) from mature bone marrow mononuclear cells [CD117-negative cells (CD117–)] from mice using the antibody to the stem cell receptor, because some of the bone marrow stem cells that express CD117+ and CD34– might generate angiogenic cytokines and differentiate into endothelial cells. The angiogenic potency of CD117+ and CD117– cells was investigated in vitro and in vivo. Significantly higher levels of VEGF were secreted from the CD117+ cells than from the CD117– cells ( P < 0.001). Most of the CD117– cells died, but the CD117+ cells grew well and differentiated into endothelial cells within 14 days of culture. The CD117+ cells survived and were incorporated in microvessels within 14 days of being implanted into the ischemic hindlimbs of mice, but the CD117– cells did not. The microvessel density and blood perfusion of the ischemic hindlimbs were significantly higher in the CD117+ cell-implanted mice than in the CD117– cell-implanted mice ( P < 0.01). The microvessel density in ischemic hindlimbs was also significantly higher in the CD117+ cell-implanted mice than in the total bone marrow cell-implanted mice ( P < 0.05). Thus CD117+ stem cells play a key role in the therapeutic angiogenesis induced by bone marrow cell implantation.
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Taylor, Mary Love, Hiba I. Dada, Hannah Florian, Paul Kelly Marcom, Carey K. Anders, Leylah Drusbosky, and Jeremy Meyer Force. "Identification of pathogenic CDK12 alterations in cell-free DNA (cfDNA) from patients with breast cancer." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): 1028. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.1028.
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1028 Background: Cyclin dependent kinase 12 ( CDK12) has both tumor suppressive and proto-oncogenic potential in metastatic breast cancers (MBC). CDK12 may be an important biomarker and target in MBC. However, a comprehensive genomic analysis of CDK12 alterations from cfDNA in MBC has not been investigated and the genomic impact of CDK12 alterations across the MBC spectrum is unknown. The purpose of this study was to identify the incidence of CDK12 genomic alterations occurring in cfDNA from patients with MBC and elucidate which CDK12 alterations may impact CDK12 kinase activity. Methods: We queried 13,070 MBC samples from the Guardant Health database between April 2019 – November 2020 to identify the incidence of CDK12 alterations detected in cfDNA. We classified each alteration type as: missense mutations, indels, or truncations. Amino acid changes occurring at conserved regions across multiple species were identified. Three-dimensional biochemical in silico analyses with ChimeraX were used to determine which CDK12 alterations may impact CDK12 kinase activity. To gain further biologic insights into CDK12 altered MBC we made associations with CDK12 alterations and co-occurring mutated genes. Results: Nonsynonymous CDK12 alterations from the Guardant Health database were found in 317 samples from a cohort of 13,070 patients indicating an overall incidence of 2.43%. Alterations included: 239 (75.4%) missense mutations; 26 (8.2%) indels; and 52 (16.4%) truncations. We identified 62 alterations within the kinase domain with all occurring at highly conserved regions across species. The most frequent hotspot mutation identified was I76M/T, occurring in 11 unique breast cancers. Three-dimensional analyses indicate that CDK12 alterations within the hinge, HRD, DFG, catalytic spine, and regulatory spine may impact CDK12 kinase activity. The significantly co-occurring mutations from the Guardant Health breast cancer database in samples with CDK12 alterations were ARID1A, APC, RB1, and PTEN. Conclusions: A modest incidence of CDK12 genomic alterations occur in cfDNA from patients with breast cancer. Novel somatic alterations in CDK12 were identified from Guardant Health that were not detected in the public domain. A portion of these occurred at highly conserved regions across species suggesting these specific CDK12 mutations may impact CDK12 kinase expression and be actionable therapeutic targets in breast cancers. Three dimensional analyses of the CDK12 gene further illustrate which specific alterations may induce CDK12 kinase expression or lead to inactivation. Co-occurring mutations reveal a unique genotype associated with CDK12 alterations that may play a biologic role in CDK12-mediated breast cancer pathogenesis. Preclinical studies to determine the prognostic and therapeutic implication of CDK12 alterations in MBC are warranted.
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Park, Shin Young, Ki Yun Kim, Do Youn Jun, Su-Kyeong Hwang, and Young Ho Kim. "G1 Cell Cycle Arrest and Extrinsic Apoptotic Mechanisms Underlying the Anti-Leukemic Activity of CDK7 Inhibitor BS-181." Cancers 12, no. 12 (December 19, 2020): 3845. http://dx.doi.org/10.3390/cancers12123845.
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In vitro antitumor activity of the CDK7 inhibitor BS-181 against human T-ALL Jurkat cells was determined. Treatment of Jurkat clones (JT/Neo) with BS-181 caused cytotoxicity and several apoptotic events, including TRAIL/DR4/DR5 upregulation, c-FLIP down-regulation, BID cleavage, BAK activation, ΔΨm loss, caspase-8/9/3 activation, and PARP cleavage. However, the BCL-2-overexpressing Jurkat clone (JT/BCL-2) abrogated these apoptotic responses. CDK7 catalyzed the activating phosphorylation of CDK1 (Thr161) and CDK2 (Thr160), and CDK-directed retinoblastoma phosphorylation was attenuated in both BS-181-treated Jurkat clones, whereas only JT/BCL-2 cells exhibited G1 cell cycle arrest. The G1-blocker hydroxyurea augmented BS-181-induced apoptosis by enhancing TRAIL/DR4/DR5 upregulation and c-FLIP down-regulation. BS-181-induced FITC–annexin V-positive apoptotic cells were mostly in the sub-G1 and G1 phases. BS-181-induced cytotoxicity and mitochondrial apoptotic events (BAK activation/ΔΨm loss/caspase-9 activation) in Jurkat clones I2.1 (FADD-deficient) and I9.2 (caspase-8-deficient) were significantly lower than in A3 (wild-type). Exogenously added recombinant TRAIL (rTRAIL) markedly synergized BS-181-induced apoptosis in A3 cells but not in normal peripheral T cells. The cotreatment cytotoxicity was significantly reduced by the DR5-blocking antibody but not by the DR4-blocking antibody. These results demonstrated that the BS-181 anti-leukemic activity is attributed to extrinsic TRAIL/DR5-dependent apoptosis preferentially induced in G1-arrested cells, and that BS-181 and rTRAIL in combination may hold promise for T-ALL treatment.
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Hupe, Marie C., Anne Offermann, Finn Becker, Vincent Joerg, Wenzel Vogel, Johannes Braegelmann, Sven Perner, and Axel Stuart Merseburger. "Targeting mediator subunits CDK8/CDK19 for treatment of advanced prostate cancer." Journal of Clinical Oncology 37, no. 7_suppl (March 1, 2019): 152. http://dx.doi.org/10.1200/jco.2019.37.7_suppl.152.
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152 Background: The mediator complex plays a pivotal role in the regulation of gene transcription. The subunits CDK8 and CDK19 are overexpressed in advanced prostate cancer (PCa) and promote migration and invasion, as shown previously by our group. The aim of this study was to analyze if CDK8/CDK19 inhibition can impede PCa progression. Methods: Immunohistochemistry for CDK8 and CDK19 was performed on a large PCa cohort (376 radical prostatectomy (RP) specimens, 39 locally advanced tumors, 32 lymph node metastases, 24 distant metastases, 73 benign prostatic specimens). PCa cell lines (DU145, PC3) were treated with CDK8/CDK19 small molecule inhibitors followed by MTT assay, wound healing assay, and Western Blot for epithelial/mesenchymal markers. Results: CDK19 is higher expressed in primary PCa vs. benign specimens and locally advanced PCa/distant metastases vs. primary PCa. CDK8 and CDK19 are higher expressed in castration-resistant specimens vs. hormone-naive specimens. CDK19 significantly correlates with grade group in RP specimens. Biochemical recurrence free survival rates are lower for patients with high vs. medium vs. no/low CDK19 expression in the tumor. CDK19 predicts recurrence free survival independently from grade group and PSA. CDK8/CDK19 inhibition results in decreased migration, while there is no effect on proliferation. Mesenchymal markers are reduced while epithelial markers are induced following CDK8/CDK19 inhibition. Conclusions: CDK19 qualifies as a prognostic marker predicting biochemical recurrence following RP. CDK8/CDK19 inhibition leads to decreased migratory potential and mesenchymal phenotype. CDK8/19 expression increases during progression to castration-resistance. Collectively, CDK8/CDK19 represents a new target for treatment of advanced and/or castration-resistant PCa.
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Gramatzki, D., T. Weiss, L. Hänsch, M. Silginer, E. J. Rushing, P. Roth, M. Gramatzki, M. Peipp, and M. Weller. "P10.19.B An immunotoxin targeting CD317 for the treatment of glioblastoma." Neuro-Oncology 24, Supplement_2 (September 1, 2022): ii53. http://dx.doi.org/10.1093/neuonc/noac174.184.
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Abstract Background CD317 is an interferon-inducible cell surface receptor expressed in several solid cancer types. HM1.24-ETA’ is a small immunotoxin with a CD317 single-chain variable fragment (svFv) antibody fused to a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’) that is explored as a novel therapeutic approach in CD317-expressing tumors. Material and Methods CD317 mRNA expression in human gliomas and its association with survival was analyzed using the database of the Cancer Genome Atlas (TCGA). CD317 protein levels in human gliomas were assessed by immunohistochemistry. CD317 mRNA expression was assessed by RT-PCR and CD317 protein levels by flow cytometry in 13 human glioma cell lines in vitro. Efficacy of HM1.24-ETA’ was analyzed in acute cytotoxicity assays in vitro. Finally, HM1.24-ETA’ was evaluated in the intracranial human LN-229 glioma xenograft nude mouse model after intravenous injection. Results Interrogation of the TCGA database showed that increased CD317 mRNA expression correlated with grade of malignancy among isocitrate dehydrogenase (IDH) wildtype and IDH-mutant gliomas. Enhanced CD317 mRNA expression was associated with inferior survival in glioblastoma and astrocytoma, IDH-mutant, WHO grade 4. Immunohistochemistry confirmed CD317 overexpression in human glioblastoma compared to lower grade astrocytomas. CD317 was expressed heterogeneously on mRNA and protein levels in glioma cell lines in vitro. HM1.24-ETA’ induced acute cytotoxicity in CD317-positive glioma cells in vitro. CD317 expression and susceptibility to HM1.24-ETA’-induced cell death were enhanced by interferon-β. HM1.24-ETA’ prolonged survival in the LN-229 xenograft nude mouse model. Conclusion These data define CD317 as a novel target for treatment of human gliomas with immunoconjugates.
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Tsoi, Helen, Kwong-Fai Wong, John M. Luk, and Don Staunton. "Clinical utility of CDH17 biomarker in tumor tissues and liquid biopsies for detection and prognostic staging of colorectal cancer (CRC)." Journal of Global Oncology 5, suppl (October 7, 2019): 53. http://dx.doi.org/10.1200/jgo.2019.5.suppl.53.
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53 Background: Cancer surveillance using blood-based biomarker tests allows for early cancer detection and more effective prompt treatment. Immunohistochemical staining of tumor-specific antigens can also provide valuable prognostic information and contribute to the design of treatment strategy. Our early studies and work from other investigators have identified cadherin-17 (CDH17) as a promising biomarker in both tumor tissues and liquid biopsies. CDH17 overexpression in cancer tissue was found predictive of poor prognosis in patients with gastric cancer, hepatocellular carcinoma and ovarian cancer. Plasma CDH17 was elevated in patients with gastric cancer when compared to healthy subjects. However, despite the diagnostic and prognostic value of CDH17, robust and accurate assays for CDH17 detection in liquid and tumor biopsies have yet to be developed. Methods: In order to establish CDH17 immunoassays, we have first generated a proprietary library of more than 300 antibodies against CDH17, and from this library, isolated a panel of monoclonal antibodies (mAbs) exhibiting specific and high-affinity binding to the different extracellular domains of CDH17. Results: The immunohistochemical staining of CRC tissues with one of our mAbs revealed a stepwise increase in CDH17 with the progression of cancer from early to late CRC. A sandwich-ELISA was also developed to show significantly elevated plasma CDH17 in patients with CRC when compared to healthy individuals. Like CDH17 expression in CRC tissues, plasma CDH17 also showed a gradual increase with progression to more advanced stages of CRC. The newly developed ELISA also demonstrated that the number of CDH17-containing exosomes was significantly higher in blood isolated from patients with CRC. Notably, our mAbs could also enumerate CDH17-positive circulating tumor cells (CTCs), of which the level was associated with tumor stage. Conclusions: All these findings clearly indicate the clinical utility of CDH17 in tumor tissues and liquid biopsies for detection and prognostic staging of CRC. Further validation of our proprietary CDH17 immunoassays are being conducted in multiple independent CRC cohorts.
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Cain, Chris. "Selecting CDK7." Science-Business eXchange 7, no. 28 (July 2014): 817. http://dx.doi.org/10.1038/scibx.2014.817.
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Zhang, Guizhong, Jian Cheng, Zhao Liu, Tian Deng, Funmilayo Oladunni Adeshakin, and Xiaochun Wan. "CD317-mediated cell cytoskeleton regulation as a novel means of tumor immune evasion." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 242.43. http://dx.doi.org/10.4049/jimmunol.204.supp.242.43.
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Abstract CD317 is frequently overexpressed in various human cancers. Although our previous study had demonstrated its pro-proliferative role in HCC development, the functions of CD317 in tumorigenesis, especially in tumor immune evasion, remain unclear. Herein we report that CD317 protects cancer cells from immune cell-mediated cytotoxicity. CD317 knockdown in cancer cells affects neither cell-cell conjugation nor effector cell activation, but markedly increases the vulnerability of cancer cells by which renders cancer cells more sensitive to YTS- or CAR-modified NK cell-mediated cytotoxicity. This sensitivity alteration cannot be restored by adding recombinant CD317 extracellular domain (317ECD) protein, excluding the possibility that CD317 acts as a traditional immune checkpoint protein that provides inhibitory signaling to effector cells. Mechanically, immunoprotective function of CD317 is mainly dependent on the interaction with RICH2, a known CD317-binding partner that is involved in the cytoskeleton regulation. Knocking down RICH2 significantly rendered CD317 incapable of protecting cancer cells from immune cell-mediated killing, suggesting CD317 facilitates tumor immune evasion by regulating tumor cell cytoskeleton to confront immune killing. Taken together, our findings reveal a novel means of tumor immune evasion and suggest CD317 as an attractive target for improving the efficiency of cancer immunotherapy.
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Peng, Fang, Chuansheng Yang, Yanan Kong, Xiaojia Huang, Yanyu Chen, Yangfan Zhou, Xinhua Xie, and Peng Liu. "CDK12 Promotes Breast Cancer Progression and Maintains Stemness by Activating c-myc/β -catenin Signaling." Current Cancer Drug Targets 20, no. 2 (February 11, 2020): 156–65. http://dx.doi.org/10.2174/1568009619666191118113220.
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Background: CDK12 is a promising therapeutic target in breast cancer with an effective ability of maintaining cancer cell stemness. Objective: We aim to investigate the mechanism of CDK12 in maintaining breast cancer stemness. Methods: CDK12 expression level was accessed by using RT-qPCR and IHC. CDK12-altered breast cancer cell lines MDA-MB-231-shCDK12 and SkBr-3-CDK12 were then established. CCK8, colony formation assays, and xenograft model were used to value the effect of CDK12 on tumorigenicity. Transwell assay, mammosphere formation, FACS, and lung metastasis model in vivo were determined. Western blot further characterized the mechanism of CDK12 in breast cancer stemness through the c-myc/β-catenin pathway. Results: Our results showed a higher level of CDK12 exhibited in breast cancer samples. Tumor formation, cancer cell mobility, spheroid forming, and the epithelial-mesenchymal transition will be enhanced in the CDK12high group. In addition, CDK12 was associated with lung metastasis and maintained breast cancer cell stemness. CDK12high cancer cells presented higher tumorigenicity and a population of CD44+ subset compared with CDK12low cells. Our study demonstrated c-myc positively expressed with CDK12. The c-myc/β-catenin signaling was activated by CDK12, which is a potential mechanism to initiate breast cancer stem cell renewal and may serve as a potential biomarker of breast cancer prognosis. Conclusion: CDK12 overexpression promotes breast cancer tumorigenesis and maintains the stemness of breast cancer by activating c-myc/β-catenin signaling. Inhibiting CDK12 expression may become a potential therapy for breast cancer.
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Larochelle, Stéphane, Karl A. Merrick, Marie-Emilie Terret, Lara Wohlbold, Nora M. Barboza, Chao Zhang, Kevan M. Shokat, Prasad V. Jallepalli, and Robert P. Fisher. "Requirements for Cdk7 in the Assembly of Cdk1/Cyclin B and Activation of Cdk2 Revealed by Chemical Genetics in Human Cells." Molecular Cell 25, no. 6 (March 2007): 839–50. http://dx.doi.org/10.1016/j.molcel.2007.02.003.
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Hänsch, L., M. Peipp, R. Myburgh, M. Silginer, T. Weiss, D. Gramatzki, F. Vasella, M. Manz, M. Weller, and P. Roth. "PL03.3.A Development and characterization of CD317-specific CAR T cells as an innovative immunotherapeutic strategy against glioblastoma." Neuro-Oncology 23, Supplement_2 (September 1, 2021): ii2. http://dx.doi.org/10.1093/neuonc/noab180.005.
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Abstract BACKGROUND Due to the limited success of existing therapies for gliomas, innovative therapeutic options are urgently needed. Chimeric antigen receptor (CAR) T cell therapy has been successful in patients with hematological malignancies. However, using this treatment against solid tumors such as glioblastomas is more challenging. Here, we generated CAR T cells targeting the transmembrane protein CD317 (BST-2, HM1.24) which is overexpressed in glioma cells and may therefore serve as a novel target antigen for CAR T cell-based immunotherapy. MATERIAL AND METHODS CAR T cells targeting CD317 were generated by lentiviral transduction of human T cells from healthy donors. The anti-glioma activity of CD317-CAR T cells was determined in lysis assays using different glioma target cell lines with varying CD317 expression levels. The efficiency of CD317-CAR T cells to control tumor growth in vivo was evaluated in clinically relevant orthotopic xenograft glioma mouse models. RESULTS We created a second-generation CAR construct targeting CD317 and observed strong anti-glioma activity of CD317-CAR T cells in vitro. Glioma cells with a CRISPR/Cas9-mediated CD317 knockout were resistant to CD317-specific CAR T cells, demonstrating their target antigen-specificity. Since CD317 is also expressed by T cells, transduction with a CD317-directed CAR resulted in fratricide of the transduced T cells. Silencing of CD317 in CAR T cells by integrating a specific shRNA into the CAR vector significantly increased the viability, proliferation and cytotoxic function of the CAR T cells. Importantly, intratumoral treatment with CD317-CAR T cells prolonged the survival and cured a significant fraction of glioma-bearing nude mice. CONCLUSION We demonstrate strong CD317-specific anti-tumor activity of CD317-CAR T cells against various glioma cell lines in vitro and in xenograft glioma models in vivo. These data lay a scientific basis for the subsequent evaluation of this therapeutic strategy in clinical neuro-oncology.
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Tun, Nay Min, and Gina M. Villani. "Predictive value of KIT immunohistochemical staining for KIT mutations in patients with gastrointestinal stromal tumors (GIST): A systematic review." Journal of Clinical Oncology 30, no. 4_suppl (February 1, 2012): 30. http://dx.doi.org/10.1200/jco.2012.30.4_suppl.30.
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30 Background: Immunohistochemical staining for KIT (CD117) is used as part of diagnostic tool for GIST. Approximately 95% of GIST are known to be positive for CD117. Studies have shown that not all CD117-positive GIST carry KIT mutations nor does CD117 negativity rule out KIT or PDGFRA mutations. We performed a systematic review to investigate the positive and negative predictive values of KIT immunostaining for KIT mutations in GIST. Methods: A Medline search of the MeSH terms “KIT mutation” and “GIST” and “prevalence” yielded 54 articles from year 1999 to 2011. English language studies on GIST with data available for KIT immunostaining as well as KIT gene mutations were included. Studies lacking data on either CD117 positivity or KIT mutations were excluded. Immunostaining was done using Dako polyclonal rabbit antibody or PKCh monoclonal mouse antibody in eligible studies. Denaturing high-pressure liquid chromatography (DHPLC) was used to screen mutations in majority of eligible studies, and ABI Prism Genetic Analyzer to sequence DNA. Results: 6 studies including 708 CD117-positive GIST patients were eligible for analysis of positive predictive value of KIT immunostaining for KIT mutations and 5 studies including 47 CD117-negative GIST patients for negative predictive value. Many studies were excluded due to insufficient data on CD117 positivity or KIT mutational analysis. 72.8% of CD117-positive GIST tumors carried KIT mutations (exon 11 62.2%, exon 9 10.2%, exon 13 1.8% and exon 17 1%), and 4.1% harbored PDGFRA mutations. In contrast, 74.5% of CD117-negative GIST tumors did not have KIT mutations. 25.5% of CD117-negative GIST harbored KIT exon 11 mutations (no mutations in exon 9, 13 or 17 were observed). In addition, 30% of CD117-negative GIST were found to have PDGFRA mutations. Conclusions: PDGFRA mutations were found to be 7 times more common in CD117-negative than in CD117-positive GIST (30% vs. 4.1%). Over half (55.5%) of CD117-negative GIST carried KIT exon 11 or PDGFRA mutations. We recommend mutational analysis in all tumors clinically suspected of GIST and that are CD117-negative for two reasons: 1. in order to make a diagnosis, and 2. to help tailor therapy.
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Yang, Bikang, Jing Chen, and Yincheng Teng. "CDK12 Promotes Cervical Cancer Progression through Enhancing Macrophage Infiltration." Journal of Immunology Research 2021 (February 11, 2021): 1–14. http://dx.doi.org/10.1155/2021/6645885.
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Cervical cancer (CC) is a commonly diagnosed and primary consideration of cancer patient death in female reproductive system malignancy. Cyclin-dependent kinase 12 (CDK12), as a transcription-associated CDK, plays important roles in tumor-promoting behaviors, whereas the underlying mechanisms of CDK12 in CC progression are still obscure. In this report, we investigated the role of CDK12 in cervical cancer. The current study identified CDK12 mRNA and protein expression remarkably upregulated in CC patients. Upregulated CDK12 was closely associated with CC progression and poor prognosis. In vitro and in vivo functional experiments showed that knockdown of CDK12 inhibited cancer cell proliferation and colony formation and promoted apoptosis. Further investigations demonstrated that CDK12 regulated the immune microenvironment to facilitate the progression of CC cells by promoting macrophage infiltration. Meanwhile, we first demonstrated that nuclear import of CDK12 is mediated by TNPO1 and might be a new therapeutic target in oncology. Collectively, this study pointed out the potential of CDK12 to serve as a novel therapeutic target in restricting CC proliferation and cell cycle process through promoting macrophage infiltration.
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Yang, L. L., L. Wu, G. T. Yu, W. F. Zhang, B. Liu, and Z. J. Sun. "CD317 Signature in Head and Neck Cancer Indicates Poor Prognosis." Journal of Dental Research 97, no. 7 (February 27, 2018): 787–94. http://dx.doi.org/10.1177/0022034518758604.
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Targeted therapy using monoclonal antibodies (mAbs) has emerged as a widely used form of immunotherapy in head and neck squamous cell carcinoma (HNSCC). Membrane-associated glycoprotein CD317 has been preferentially overexpressed by multiple myeloma cells, and its humanized mAb has been previously used in clinical trials. However, overexpression of CD317 in HNSCC and its correlation with tumor immunity is still uncertain. Here, the immunoreactivity of CD317 was detected in human HNSCC tissue microarrays, which contained 43 oral mucosa samples, 48 dysplasia samples, and 165 primary HNSCC. We found that CD317 expression was up-regulated in HNSCC tumor cells, and the CD317 expression level was independent of the histological grade, tumor size, and lymph node metastasis. Moreover, Kaplan–Meier survival curve analysis showed that patients with high expression of CD317 had a poor prognosis compared with patients with low expression. Furthermore, CD317 overexpression in HNSCC was correlated with immune checkpoint molecules PD-L1, B7-H3, and B7-H4 and tumor-associated macrophage markers (CD68 and CD163). We also observed that CD317 was overexpressed in immunocompetent mouse HNSCC tissue compared with normal tissue. Taken together, our findings demonstrate that CD317 overexpression indicates poor prognosis and is correlated with immune-related components in this patient cohort. CD317 may serve as a potential target for effective immunotherapy of HNSCC.
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Gramatzki, Dorothee, Emese Szabo, Martin Gramatzki, Matthias Peipp, and Michael Weller. "Targeting of CD317 by the immunotoxin HM1.24-ETA’ to allow immunotherapy in glioblastoma patients." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e13560-e13560. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13560.
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e13560 Background: Glioblastoma is the most common primary malignant brain tumor with a poor prognosis. CD317 (HM1.24) is a transmembrane protein and may exist in differently spliced variants. It is highly expressed on plasma cells in multiple myeloma, as well as in certain solid tumor types. While several antibody drug conjugates are already in clinical practice, small immunotoxins with a different intracellular mode of action are only established in hairy cell leukemia. The immunotoxin HM1.24-ETA’ protein is a CD317 single chain Fv (scFv) antibody fused to a truncated version of Pseudomonas aeruginosa exotoxin A (ETA’). Methods: In vivo CD317 mRNA expression in human glioma of different grades and survival probabilities of glioblastoma patients based on CD317 mRNA expression were analyzed using the database of the Cancer Genome Atlas network (TCGA). CD317 protein expression was analyzed by immunohistochemistry in a human tissue microarray (TMA). In vitro CD317 mRNA expression was assessed by RT-PCR and CD317 protein levels by flow cytometry in several human glioblastoma cell lines. A cytotoxicity assay after treatment with HM1.24-ETA’ immunotoxin was performed in human glioblastoma cell lines. Results: Data on mRNA expression from the TCGA database demonstrated, that CD317 was upregulated in human glioblastomas compared to lower grade gliomas. In the group of glioblastoma patients increased CD317 mRNA expression was associated with decreased probability of survival ( p< 0.001). CD317 protein levels correlated directly with the tumor grade of astrocytic gliomas in the TMA. CD317 was expressed heterogeneously on mRNA and protein levels in the tested cell-lines in vitro. HM1.24-ETA’ induced cytotoxicity in CD317-positive glioblastoma cells in a concentration-dependent manner. Animal experiments currently performed suggest activity in glioblastoma xenografted mice. Conclusions: These data highlight CD317 as an interesting target antigen and HM1.24-ETA’ immunotoxin as a strategy for immunotherapy of glioblastoma patients.
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Li, Juan, Shaokai Luo, Guocai Zhang, Wende Hong, and Xiuzhen Tong. "Expression of the CD117 Antigen on Multiple Myeloma and Its Significance." Blood 104, no. 11 (November 16, 2004): 4867. http://dx.doi.org/10.1182/blood.v104.11.4867.4867.
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Abstract BACKGROUND & OBJECTIVE: Leukocyte differentiation antigen CD117 is one of the targets that tyrosine kinase selective inhibitors work on. CD117, whether the cell surface is expressed and the quality of its expression, is highly correlated with the tyrosine kinase selective inhibitors. And whether Multiple myeloma (MM) cells express CD117 and its expression quality is not reported domestic yet but only several reported overseas. In this study, CD117 expressed in MM cells is evaluated, which provide an theoretical evidence for tyrosine kinase selective inhibitors used in the MM, meanwhile, the value of the CD117 expressed in MM cells is estimated. METHODS:CD117,CD56,CD54 were measured by three -color flow cytometry with CD45/SSC gating strategy. RESULTS:Of 48 patients with MM, 17(35.5%) CD117 expression was positive in myeloma cells, and CD56, CD54 expression was positive in 39(81.2%), 48(100.0%), respectively. CD117 expression in myeloma cells was low compared with CD56, CD54 in 48 patients with MM; CD117 positive expression showed a positive correlation with myeloma cells in the bone marrow; CD117 positive in IgG type of MM was 64%, higher than other types such as light chain or IgA.CD117 positive showed no significant difference in different stage, untreated, relapsed and refractory patients (P>0.05, P>0.01); In untreated MM patient, chemotherapy of VAD in CD117 positive patient, the effectiveness was 71.4%, compared with the reaction rate 66.7% in CD117 negative, showed no significant difference (P>0.05). CONCLUSIONS:CD117 </SU
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Zhou, Jun, Xiaoqun Yang, Luting Zhou, Peipei Zhang, and Chaofu Wang. "Combined Immunohistochemistry for the “Three 7” Markers (CK7, CD117, and Claudin-7) Is Useful in the Diagnosis of Chromophobe Renal Cell Carcinoma and for the Exclusion of Mimics: Diagnostic Experience from a Single Institution." Disease Markers 2019 (October 13, 2019): 1–9. http://dx.doi.org/10.1155/2019/4708154.
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Background. There is a morphological overlap among renal epithelial tumors, particularly chromophobe renal cell carcinoma (CHRCC), clear cell renal cell carcinoma (CCRCC), renal oncocytoma (RO), and papillary renal cell carcinoma (PRCC). Discriminating between these tumors is important but sometimes challenging. This study is aimed at evaluating the clinical usefulness of the combined immunochemistry for the “three 7” markers (CK7, CD117, and Claudin-7) to distinguish chromophobe renal cell carcinoma from these mimics. Methods. Immunochemical staining for CK7, CD117, and Claudin-7 was performed in 68 CHRCCs, 199 CCRCCs, 32 ROs, and 30 PRCCs. Fluorescence in situ hybridization (FISH) was performed in some cases to exclude CCRCC and PRCC. The sensitivity (SE) and specificity (SP) for CHRCC as well as the immunoreactivity of each marker and their combinations were statistically evaluated. Results. High positive rates for CK7 (94%), CD117 (87%), Claudin-7 (94%), and their combinations (CK7+CD117, 79%; CK7+Claudin-7, 88%; CD117+Claudin-7, 82%; CK7+CD117+Claudin-7, 76%) were observed in CHRCC compared to those in CCRCC, RO, and PRCC, with increasingly higher SP when combinations of the “three 7” markers were applied (CK7, 0.80; CD117, 0.82; Claudin-7, 0.78; CK7+CD117, 0.95; CK7+Claudin-7, 0.97; CD117+Claudin-7, 0.97; CK7+CD117+Claudin-7, 1). Conclusion. CK7, CD117, and Claudin-7 are frequently expressed in CHRCC with high specificity. We recommend the routine use of these 3 markers as a routine panel when making a differential diagnosis of CHRCC and excluding other mimics.
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Goh, Kee C., Wai C. Ong, Changyong Hu, Ai L. Liang, Walter Stunkel, Yong C. Tan, Kanda Sangthongpitag, et al. "SB1317, a Potent and Orally Active FLT3-CDK Inhibitor with High Anti-Tumor Efficacy in Models of Hematological Malignancies." Blood 110, no. 11 (November 16, 2007): 1593. http://dx.doi.org/10.1182/blood.v110.11.1593.1593.
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Abstract FLT3 is the most common mutated gene in acute myeloid leukemia (AML), and FLT3 mutations are strongly correlated with poor prognosis. Small-molecule inhibitors of FLT3 kinase have been evaluated in clinical trials but with limited success due to the emergence of activating mutants other than FLT3. CDK1, 2, 4 and 6 are well-established anti-cancer targets due to their direct role in cell cycle control. CDK7 and 9 are transcriptional CDKs that are emerging anti-cancer targets as a result of recent advances in the understanding of their effects on apoptotic regulators. Recent evidence suggests that the combined targeting of CDK1, 2 and 9 enhances apoptotic killing of tumor cells. Most CDK active compounds currently in clinical development do not target all of these CDKs and have poor pharmacokinetic/pharmacodynamic properties. Compounds targeting these CDKs, in addition to FLT3, may be more effective in AML and also be effective against other hematological as well as solid tumors. SB1317 is a novel potent inhibitor of FLT3 kinase (IC50 = 45 nM) and CDK2 (IC50 = 11 nM). The CDK spectrum also includes potent inhibition of CDK1 and 9 (IC50 = 19 and 10 nM respectively). SB1317 inhibits proliferation of a broad panel of tumor cell lines, and is particularly potent against the mutant FLT3-dependent AML cell line, MV4-11 (proliferation IC50 = 18 nM). SB1317 reduced phospho-Rb and phospho-FLT3 levels in MV4-11 cells in a dose-dependent manner. It induced apoptosis in MV4-11 cells as well as in primary AML cells from patients. The pharmaceutical, metabolic and pharmacokinetic properties of SB1317 render it amenable to oral dosing. In a nude mouse subcutaneous model of AML (MV4-11), SB1317 induced complete tumor regression after oral daily dosing (40 mg/kg for 21 days). In an orthotopic model of AML (HL60), SB1317 significantly prolonged median survival time (59 days versus 40 days) after treatment at 100 mg/kg p.o. 2d on/5d off. It also showed significant anti-tumor activity in a nude mouse model of B-cell lymphoma (Ramos) with a tumor growth inhibition of 63% (15 mg/kg i.p. 5d on/5d off). These results demonstrate the therapeutic potential of this novel kinase inhibitor for the treatment of hematological malignancies.
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Chalfant, Victor, Carlos Riveros, Sanjeev Shukla, Teruko Osumi, and K. Balaji. "Abstract 1622: Signaling of cyclin-dependent kinase 12 (CDK12) in prostate cancer cell lines." Cancer Research 82, no. 12_Supplement (June 15, 2022): 1622. http://dx.doi.org/10.1158/1538-7445.am2022-1622.
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Abstract Cyclin-dependent kinase 12 (CDK12) belongs to the cyclin-dependent kinase (CDK) family of serine/threonine protein kinases. In contrast to CDKs which promote cell cycle progression, CDK12 is a transcriptional regulator of various cellular functions, most importantly cellular response to DNA damage and stress. Genomic alterations in CDK12 have been detected in up to 7% of patients with metastatic castration-resistant prostate cancer (CRPC). Phosphoproteomic studies have revealed that Protein Kinase D1 (PrKD1), another member of the serine/threonine kinase family, is the only kinase known to phosphorylate CDK12 at serine 681 (s681) and serine 685 (s685). While there is an increasing body of literature on CDK12 downstream signaling, there is almost no published data on upstream effectors or regulation of CDK12. Using site-directed mutagenesis, we generated CDK12 s681a and s681a+s685 mutants and transfected them into stable C4-2 and C4-2-PrKD1 cell lines to evaluate the interaction between CDK12 and PrKD1. Immunoprecipitation and immunoblotting revealed that endogenous CDK12 is stable in C4-2 cells with known levels of PrKD1 expression. However, stable transfection of C4-2-PrKD1 cells with wild-type CDK12 resulted in the degradation of nascent CDK12, whereas the non-phosphorylated mutants (s681 and s681+s685) remained stable. Our results suggest that PrKD1-mediated phosphorylation of s681and/or s685 residues affect CDK12 stability in C4-2-PrKD1 cells. We confirmed this finding by treating C4-2-PrKD1 cells with MG-132, a 26 S proteasome inhibitor, and repeating the experiment. Proteasomal inhibition rescued nascent transfected CDK12 levels even after PrKD1 phosphorylation. Our study demonstrates that CDK12 degradation might be mediated by PrKD1 phosphorylation at s681 and/or s685. Because PrKD1 and CKD12 are dysregulated across cancers, the results herein presented have potential implications for the treatment of not only prostate cancer but also other human malignancies. Citation Format: Victor Chalfant, Carlos Riveros, Sanjeev Shukla, Teruko Osumi, K Balaji. Signaling of cyclin-dependent kinase 12 (CDK12) in prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1622.
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Fujiwara, Kentaro, Atsushi B. Tsuji, Hitomi Sudo, Aya Sugyo, Hiroki Akiba, Hiroko Iwanari, Osamu Kusano-Arai, et al. "111In-labeled anti-cadherin17 antibody D2101 has potential as a noninvasive imaging probe for diagnosing gastric cancer and lymph-node metastasis." Annals of Nuclear Medicine 34, no. 1 (October 12, 2019): 13–23. http://dx.doi.org/10.1007/s12149-019-01408-y.
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Abstract Objective Cadherin-17 (CDH17) is a transmembrane protein that mediates cell–cell adhesion and is frequently expressed in adenocarcinomas, including gastric cancer. CDH17 may be an effective diagnostic marker for the staging of gastric cancer. Here, we developed an 111In-labeled anti-CDH17 monoclonal antibody (Mab) as an imaging tracer and performed biodistribution and single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies using mice with CDH17-positive gastric cancer xenografts. CDH17 expression in gastric cancer specimens was also analyzed. Methods The cross-reactivity and affinity of our anti-CDH17 Mab D2101 was evaluated by surface plasmon resonance analysis and cell enzyme-linked immunosorbent assay, respectively. Biodistribution and SPECT/CT studies of 111In-labeled D2101 (111In-D2101) were performed. CDH17 expression in gastric cancer specimens was evaluated by immunohistochemistry. Results Surface plasmon resonance analysis revealed that D2101 specifically recognizes human CDH17, but not murine CDH17. The affinity of D2101 slightly decreased as a result of the radiolabeling procedures. The biodistribution study revealed high uptake of 111In-D2101 in tumors (maximum, 39.2 ± 9.5% ID/g at 96 h postinjection), but low uptake in normal organs, including the stomach. Temporal SPECT/CT imaging with 111In-D2101 visualized tumors with a high degree of tumor-to-nontumor contrast. Immunohistochemical analysis revealed that, compared with HER2, which is a potential marker of N-stage, CDH17 had a higher frequency of positivity in specimens of primary and metastatic gastric cancer. Conclusion Our 111In-anti-CDH17 Mab D2101 depicted CDH17-positive gastric cancer xenografts in vivo and has the potential to be an imaging probe for the diagnosis of primary lesions and lymph-node metastasis in gastric cancer.
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Ahmed, Rehana L., Daniel P. Shaughnessy, Todd P. Knutson, Rachel I. Vogel, Khalil Ahmed, Betsy T. Kren, and Janeen H. Trembley. "CDK11 Loss Induces Cell Cycle Dysfunction and Death of BRAF and NRAS Melanoma Cells." Pharmaceuticals 12, no. 2 (April 2, 2019): 50. http://dx.doi.org/10.3390/ph12020050.
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Cyclin dependent kinase 11 (CDK11) is a protein kinase that regulates RNA transcription, pre-mRNA splicing, mitosis, and cell death. Targeting of CDK11 expression levels is effective in the experimental treatment of breast and other cancers, but these data are lacking in melanoma. To understand CDK11 function in melanoma, we evaluated protein and RNA levels of CDK11, Cyclin L1 and Cyclin L2 in benign melanocytes and BRAF- as well as NRAS-mutant melanoma cell lines. We investigated the effectiveness of reducing expression of this survival kinase using RNA interference on viability, clonal survival, and tumorsphere formation in melanoma cell lines. We examined the impact of CDK11 loss in BRAF-mutant melanoma on more than 700 genes important in cancer signaling pathways. Follow-up analysis evaluated how CDK11 loss alters cell cycle function in BRAF- and NRAS-mutant melanoma cells. We present data on CDK11, CCNL1 and CCNL2 mRNA expression in melanoma patients, including prognosis for survival. In sum, we found that CDK11 is necessary for melanoma cell survival, and a major impact of CDK11 loss in melanoma is to cause disruption of the cell cycle distribution with accumulation of G1- and loss of G2/M-phase cancer cells.
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Wang, Dong, Bethany Veo, Angela Pierce, Sujatha Venkataraman, and Rajeev Vibhakar. "MEDB-81. Combined inhibition of CDK11 and EZH2 results in regression of MYC-amplified medulloblastoma." Neuro-Oncology 24, Supplement_1 (June 1, 2022): i125. http://dx.doi.org/10.1093/neuonc/noac079.455.
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Abstract We explored an shRNA library screen on 20 cyclin-dependent kinases to establish cyclin-dependent kinase 11 (CDK11) as a critical mediator in MYC-driven medulloblastoma. The effect and molecular mechanism of CDK11 in the proliferation and growth of medulloblastoma were investigated in vitro. Pharmaceutic inhibitors and genetic depletion of CDK11 resulted in cessation of tumor growth in xenograft mouse models. Through combination chemical screening, we identified that 5-FU enhanced the apoptosis which induced by inhibition of CDK11 in medulloblastoma cells. In addition, we found CDK11 is a significant candidate kinase participating in the negative control of Wnt/b-catenin signaling. Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes through activation of transmembrane Frizzled (FZD) receptors which is suppressed by H3K27Me3. RNASeq and cut&run revealed that Cdk11 and mediator associated Cdk8 kinase regulate a common set of genes. Lack of Cdk8 and Cdk11 impaired Ezh2 recruitment and the establishment of histone H3 lysine 27 tri-methylation. We concluded that combined EZH2 and CDK8/CDK11 inhibitors treatment concurrently activated Wnt signaling may be an effective treatment for Group 3 medulloblastoma.
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Валова, Я. В., Э. Ф. Муллагалеева, Э. Т. Мингажева, Д. С. Прокофьева, А. Х. Нургалиева, Р. Р. Фаисханова, and Э. К. Хуснутдинова. "Screening for a variant of the splicing site in the CDK12 gene in patients with ovarian cancer." Nauchno-prakticheskii zhurnal «Medicinskaia genetika», no. 6(215) (June 29, 2020): 42–43. http://dx.doi.org/10.25557/2073-7998.2020.06.42-43.
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Рак яичников (РЯ) представляет собой важную проблему здравоохранения во всем мире. Данная онкопатология имеет самые высокие показатели смертности и самые низкие показатели выживаемости в течение первого года среди злокачественных опухолей женской репродуктивной системы. В связи с этим, разработка и усовершенствование методов ранней диагностики РЯ являются одной из приоритетных задач онкогинекологии. В настоящей работе представлен поиск ассоциаций варианта сайта сплайсинга c.1047-2A> G в гене CDK12 с развитием рака яичников в Республики Башкортостан. Ovarian cancer (OV) is an important public health problem worldwide. This oncopathology has the highest mortality rates and the lowest survival rates during the first year among malignant tumors of the female reproductive system. In this regard, the development and improvement of methods for the early diagnosis of cancer is one of the priority tasks of gynecological oncology. This paper presents a search for associations of a variant of the splicing site c.1047-2A> G in the CDK12 gene with the development of ovarian cancer in the Republic of Bashkortostan.
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Hupe, Marie C., Anne Offermann, Cleopatra Schreiber, Axel Stuart Merseburger, and Sven Perner. "CDK12 upregulation and adverse correlation with tumor-associated immune cell infiltrates in prostate cancer." Journal of Clinical Oncology 38, no. 6_suppl (February 20, 2020): 181. http://dx.doi.org/10.1200/jco.2020.38.6_suppl.181.
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181 Background: Biallelic loss of CDK12 has recently been identified as a novel subtype of prostate cancer (PCa). CDK12 altered PCa associates with elevated neoantigen burden and thus may be suitable for checkpoint inhibition. Up to now, data about CDK12 refer to its genetic alterations in PCa while its characterization on protein level and its association with tumor infiltrating T-cells are lacking. Methods: Immunohistochemistry (IHC) for CDK12 was performed on a PCa cohort including 74 benigns, 391 primary tumors from 222 patients, 63 locally advanced tumors, 92 lymph node (LN) metastases, and 56 distant metastases. CDK12 was categorized into negative, weak, moderate and high expression. Density of tumor associated T-cells per tumor area was assessed by IHC for CD3 and graduated into negative (<1%), slight (1-5%), weak (5-10%), moderate (10-50%) and high (>50%). Results: CDK12 significantly increases during PCa progression showing highest levels in LN and distant metastases while benign samples harbor no or weak CDK12 expression (ANOVA p<0.001). Kaplan-Meier curve reveals 5-year-biochemical recurrence free survival rates of 89.5%, 69.1%, 59.1% and 20.0% for primary tumors expressing no, weak, moderate and high CDK12 (log-rank p=0.05). High CDK12 expression significantly associates with attenuated tumor associated T-cells (p=0.009) revealing CD3 negativity in 64.7% of CDK12 high expressing tumors. Intratumoral CDK12 and density of CD3 positive T-cells correlates adversely in particular in locally advanced tumors (p=0.007). Overall, tumor associated T-cells are significantly reduced in distant metastases compared to local PCa (p<0.001). Conclusions: Our study highlights the prognostic potential of CDK12 for PCa and its overexpression in advanced tumors. Of note, CDK12 overexpressing tumors can be designated as immunologic “cold” tumors which is in line with their more aggressive phenotype. Concordantly, distant metastases show attenuated tumor associated T-cells supporting the poor response to immunotherapy.
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Alfaleh, Mohamed, Neetika Arora, Michael Yeh, Christopher de Bakker, Christopher Howard, Philip Macpherson, Rachel Allavena, et al. "Canine CD117-Specific Antibodies with Diverse Binding Properties Isolated from a Phage Display Library Using Cell-Based Biopanning." Antibodies 8, no. 1 (February 12, 2019): 15. http://dx.doi.org/10.3390/antib8010015.
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CD117 (c-Kit) is a tyrosine kinase receptor that is overexpressed in multiple dog tumors. There is 100% homology between the juxtamembrane domain of human and canine CD117, and many cancer-causing mutations occur in this region in both species. Thus, CD117 is an important target for cancer treatment in dogs and for comparative oncology studies. Currently, there is no monoclonal antibody (mAb) specifically designed to target the exposed region of canine CD117, although there exist some with species cross-reactivity. We panned a naïve phage display library to isolate antibodies against recombinant CD117 on whole cells. Several mAbs were isolated and were shown to bind recombinant canine CD117 at low- to sub-nanomolar affinity. Additionally, binding to native canine CD117 was confirmed by immunohistochemistry and by flow cytometry. Competitive binding assays also identified mAbs that competed with the CD117 receptor-specific ligand, the stem cell factor (SCF). These results show the ability of our cell-based biopanning strategy to isolate a panel of antibodies that have varied characteristics when used in different binding assays. These in vitro/ex vivo assessments suggest that some of the isolated mAbs might be promising candidates for targeting overexpressed CD117 in canine cancers for different useful applications.
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Ludwig, Marion, Anita Tölk, Anna Skorska, Christian Maschmeier, Ralf Gaebel, Cornelia Aquilina Lux, Gustav Steinhoff, and Robert David. "Exploiting AT2R to Improve CD117 Stem Cell Function In Vitro and In Vivo - Perspectives for Cardiac Stem Cell Therapy." Cellular Physiology and Biochemistry 37, no. 1 (2015): 77–93. http://dx.doi.org/10.1159/000430335.
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Background/Aims: CD117+ stem cell (SC) based therapy is considered an alternative therapeutic option for terminal heart disease. However, controversies exist on the effects of CD117+ SC implantation. In particular, the link between CD117+ SC function and angiotensin-II-type-2 receptor (AT2R) after MI is continuously discussed. We therefore asked whether 1) AT2R stimulation influences CD117+ SC properties in vitro and, 2) which effects can be ascribed to AT2R stimulation in vivo. Methods: We approached AT2R stimulation with Angiotensin II while simultaneously blocking its opponent receptor AT1 with Losartan. CD117 effects were dissected using a 2D-Matrigel assay and HL-1 co-culture in vitro. A model of myocardial infarction, in which we implanted EGFP+ CD117 SC, was further applied. Results: While we found indications for AT2R driven vasculogenesis in vitro, co-culture experiments revealed that CD117+ SC improve vitality of cardiomyocytes independently of AT2R function. Likewise, untreated CD117+ SC had a positive effect on cardiac function and acted cardioprotective in vivo. Conclusions: Therefore, our data show that transient AT2R stimulation does not significantly add to the beneficial actions of CD117+ SC in vivo. Yet, exploiting AT2R driven vasculogenis via an optimized AT2R stimulation protocol may become a promising tool for cardiac SC therapy.