Transcriptional Cyclin-dependent Kinases Research Articles

CDK9 Inhibition by Dinaciclib Is a Therapeutic Vulnerability in Epithelioid Hemangioendothelioma.

There are no effective treatment options for patients with aggressive epithelioid hemangioendothelioma (EHE) driven by the TAZ-CAMTA1 (TC) fusion gene. Here, we aimed to understand the regulation of TC using pharmacologic tools and identify vulnerabilities that can potentially be exploited for the treatment of EHE. TC is a transcriptional coregulator; we hypothesized that compounds that reduce TC nuclear levels, either through translocation of TC to the cytoplasm, or through degradation, would render TC less oncogenic. TC localization was monitored using immunofluorescence in an EHE tumor cell line. Two target-selective libraries were used to identify small molecules that reduce TC localization in the nucleus. The ability of the shortlisted hits to affect cell viability, apoptosis, and tumorigenesis was also evaluated. Basal TC remained "immobile" in the nucleus; administration of cyclin-dependent kinase (CDK) inhibitors such as CGP60474 and dinaciclib (Dina) mobilized TC. "Mobile" TC shuttled between the nucleus and cytoplasm; however, it was eventually degraded through proteasomes. This dramatically suppressed the levels of TC-regulated transcripts and cell viability, promoted apoptosis, and reduced the area of metastatic lesions in the allograft model of EHE. We specifically identified that the inhibition of CDK9, a transcriptional CDK, destabilizes TC. The CDK inhibitor Dina exhibited antitumorigenic properties both in vitro and in vivo in EHE models. Dina has been rigorously tested in clinical trials and displayed an acceptable toxicity profile. Therefore, there is a potential therapeutic window for repurposing Dina for the treatment of EHE.

Small molecule inhibitors of transcriptional cyclin-dependent kinases impose HIV-1 latency, presenting "block and lock" treatment strategies.

Current antiretroviral therapy for HIV-1 infection does not represent a cure for infection as viral rebound inevitably occurs following discontinuation of treatment. The "block and lock" therapeutic strategy is intended to enforce proviral latency and durably suppress viremic reemergence in the absence of other intervention. The transcription-associated cyclin-dependent protein kinases (tCDKs) are required for expression from the 5´ HIV-1 long-terminal repeat, but the therapeutic potential of inhibiting these kinases for enforcing HIV-1 latency has not been characterized. Here, we expanded previous observations to directly compare the effect of highly selective small molecule inhibitors of CDK7 (YKL-5-124), CDK9 (LDC000067), and CDK8/19 (Senexin A), and found each of these prevented HIV-1 provirus expression at concentrations that did not cause cell toxicity. Inhibition of CDK7 caused cell cycle arrest, whereas CDK9 and CDK8/19 inhibitors did not, and could be continuously administered to establish proviral latency. Upon discontinuation of drug administration, HIV immediately rebounded in cells that had been treated with the CDK9 inhibitor, while proviral latency persisted for several days in cells that had been treated with CDK8/19 inhibitors. These results identify the mediator kinases CDK8/CDK19 as potential "block and lock" targets for therapeutic suppression of HIV-1 provirus expression.

The CDK12 inhibitor SR-4835 functions as a molecular glue that promotes cyclin K degradation in melanoma

CDK12 is a transcriptional cyclin-dependent kinase (CDK) that interacts with cyclin K to regulate different aspects of gene expression. The CDK12-cyclin K complex phosphorylates several substrates, including RNA polymerase II (Pol II), and thereby regulates transcription elongation, RNA splicing, as well as cleavage and polyadenylation. Because of its implication in cancer, including breast cancer and melanoma, multiple pharmacological inhibitors of CDK12 have been identified to date, including THZ531 and SR-4835. While both CDK12 inhibitors affect Poll II phosphorylation, we found that SR-4835 uniquely promotes cyclin K degradation via the proteasome. Using loss-of-function genetic screening, we found that SR-4835 cytotoxicity depends on a functional CUL4-RBX1-DDB1 ubiquitin ligase complex. Consistent with this, we show that DDB1 is required for cyclin K degradation, and that SR-4835 promotes DDB1 interaction with the CDK12-cyclin K complex. Docking studies and structure-activity relationship analyses of SR-4835 revealed the importance of the benzimidazole side-chain in molecular glue activity. Together, our results indicate that SR-4835 acts as a molecular glue that recruits the CDK12-cyclin K complex to the CUL4-RBX1-DDB1 ubiquitin ligase complex to target cyclin K for degradation.

Distinct states of nucleolar stress induced by anticancer drugs

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed ‘nucleolar normality score’ was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I– recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development.

Distinct states of nucleolar stress induced by anticancer drugs

Ribosome biogenesis is a vital and highly energy-consuming cellular function occurring primarily in the nucleolus. Cancer cells have an elevated demand for ribosomes to sustain continuous proliferation. This study evaluated the impact of existing anticancer drugs on the nucleolus by screening a library of anticancer compounds for drugs that induce nucleolar stress. For a readout, a novel parameter termed ‘nucleolar normality score’ was developed that measures the ratio of the fibrillar center and granular component proteins in the nucleolus and nucleoplasm. Multiple classes of drugs were found to induce nucleolar stress, including DNA intercalators, inhibitors of mTOR/PI3K, heat shock proteins, proteasome, and cyclin-dependent kinases (CDKs). Each class of drugs induced morphologically and molecularly distinct states of nucleolar stress accompanied by changes in nucleolar biophysical properties. In-depth characterization focused on the nucleolar stress induced by inhibition of transcriptional CDKs, particularly CDK9, the main CDK that regulates RNA Pol II. Multiple CDK substrates were identified in the nucleolus, including RNA Pol I– recruiting protein Treacle, which was phosphorylated by CDK9 in vitro. These results revealed a concerted regulation of RNA Pol I and Pol II by transcriptional CDKs. Our findings exposed many classes of chemotherapy compounds that are capable of inducing nucleolar stress, and we recommend considering this in anticancer drug development.

Discovery of KB-0742, a Potent, Selective, Orally Bioavailable Small Molecule Inhibitor of CDK9 for MYC-Dependent Cancers.

Transcriptional deregulation is a hallmark of many cancers and is exemplified by genomic amplifications of the MYC family of oncogenes, which occur in at least 20% of all solid tumors in adults. Targeting of transcriptional cofactors and the transcriptional cyclin-dependent kinase (CDK9) has emerged as a therapeutic strategy to interdict deregulated transcriptional activity including oncogenic MYC. Here, we report the structural optimization of a small molecule microarray hit, prioritizing maintenance of CDK9 selectivity while improving on-target potency and overall physicochemical and pharmacokinetic (PK) properties. This led to the discovery of the potent, selective, orally bioavailable CDK9 inhibitor 28 (KB-0742). Compound 28 exhibits in vivo antitumor activity in mouse xenograft models and a projected human PK profile anticipated to enable efficacious oral dosing. Notably, 28 is currently being investigated in a phase 1/2 dose escalation and expansion clinical trial in patients with relapsed or refractory solid tumors.

P10.35.A CDK12/CDK13 INHIBITION DISRUPTS A TRANSCRIPTIONAL PROGRAM CRITICAL FOR GLIOBLASTOMA SURVIVAL

Abstract BACKGROUND Glioblastoma is the most prevalent and aggressive malignant tumor of the central nervous system. Aberrant transcriptional programs are key to development and maintenance of cancer cells, and these can be exploited to target a specific cancer. Thus, targeting the transcriptional addiction specific for gliomas could offer a large therapeutic potential. METHODS A panel of patient-derived primary glioma stem cell (GSC) lines and glioblastoma organoids (GBO) were used to investigate the effect of transcriptional cyclin-dependent kinase (tCDK) inhibitors using a range of survival assays. A CRISPR/CAS9-based competition assay was applied to study the effect of genetic ablation of individual tCDKs. CUT&RUN was utilized to generate the genome-wide maps of the total RNA polymerase II (RNAPII) and phosphorylated species, and SLAM-seq was used to identify changes in steady-state and nascent transcriptome. Cell cycle analyses were done using EdU incorporation to mark S-phase cells. RESULTS We show that pharmacological inhibition or genetic ablation of the tCDKs, CDK12 and CDK13, markedly reduces the in vitro proliferation of GSCs and of ex vivo glioblastoma organoids as well as migratory capacity of GSCs. Using a xenograft mouse model, we demonstrate that CDK12/13 inhibition reduces tumor growth in vivo, which compares favorably with the existing chemotherapeutic treatments. Mechanistically, inhibition of CDK12/CDK13 leads to a genome-wide abrogation of RNAPII C-terminal Domain (CTD) phosphorylation, which in turn disrupts transcription and cell cycle progression in glioblastoma cells. CONCLUSION s CDK12/CDK13 inhibition may have a large therapeutic potential for glioblastoma treatment, both alone and in combination.

MYC up-regulation confers vulnerability to dual inhibition of CDK12 and CDK13 in high-risk Group 3 medulloblastoma

BackgroundMedulloblastoma (MB) is the most common cerebellar malignancy during childhood. Among MB, MYC-amplified Group 3 tumors display the worst prognosis. MYC is an oncogenic transcription factor currently thought to be undruggable. Nevertheless, targeting MYC-dependent processes (i.e. transcription and RNA processing regulation) represents a promising approach.MethodsWe have tested the sensitivity of MYC-driven Group 3 MB cells to a pool of transcription and splicing inhibitors that display a wide spectrum of targets. Among them, we focus on THZ531, an inhibitor of the transcriptional cyclin-dependent kinases (CDK) 12 and 13. High-throughput RNA-sequencing analyses followed by bioinformatics and functional analyses were carried out to elucidate the molecular mechanism(s) underlying the susceptibility of Group 3 MB to CDK12/13 chemical inhibition. Data from International Cancer Genome Consortium (ICGC) and other public databases were mined to evaluate the functional relevance of the cellular pathway/s affected by the treatment with THZ531 in Group 3 MB patients.ResultsWe found that pharmacological inhibition of CDK12/13 is highly selective for MYC-high Group 3 MB cells with respect to MYC-low MB cells. We identified a subset of genes enriched in functional terms related to the DNA damage response (DDR) that are up-regulated in Group 3 MB and repressed by CDK12/13 inhibition. Accordingly, MYC- and CDK12/13-dependent higher expression of DDR genes in Group 3 MB cells limits the toxic effects of endogenous DNA lesions in these cells. More importantly, chemical inhibition of CDK12/13 impaired the DDR and induced irreparable DNA damage exclusively in MYC-high Group 3 MB cells. The augmented sensitivity of MYC-high MB cells to CDK12/13 inhibition relies on the higher elongation rate of the RNA polymerase II in DDR genes. Lastly, combined treatments with THZ531 and DNA damage-inducing agents synergically suppressed viability of MYC-high Group 3 MB cells.ConclusionsOur study demonstrates that CDK12/13 activity represents an exploitable vulnerability in MYC-high Group 3 MB and may pave the ground for new therapeutic approaches for this high-risk brain tumor.

Targeting Transcriptional CDKs 7, 8, and 9 with Anilinopyrimidine Derivatives as Anticancer Agents: Design, Synthesis, Biological Evaluation and In Silico Studies.

Cyclin-dependent kinases (CDKs) are promising targets in chemotherapy. In this study, we report a series of 2-anilinopyrimidine derivatives with CDK inhibitory activity. Twenty-one compounds were synthesized and their CDK inhibitory and cytotoxic activities were evaluated. The representative compounds demonstrate potent antiproliferative activities toward different solid cancer cell lines and provide a promising strategy for the treatment of malignant tumors. Compound 5f was the most potent CDK7 inhibitor (IC50 = 0.479 µM), compound 5d was the most potent CDK8 inhibitor (IC50 = 0.716 µM), and compound 5b was the most potent CDK9 inhibitor (IC50 = 0.059 µM). All the compounds satisfied the Lipinski's rule of five (molecular weight < 500 Da, number of hydrogen bond acceptors <10, and octanol-water partition coefficient and hydrogen bond donor values below 5). Compound 5j is a good candidate for lead optimization because it has a non-hydrogen atom (N) of 23, an acceptable ligand efficiency value of 0.38673, and an acceptable ligand lipophilic efficiency value of 5.5526. The synthesized anilinopyrimidine derivatives have potential as anticancer agents.

Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

Abstract 299: Large tandem duplications in cancer resulting from transcription and DNA replication collisions

Abstract Somatic structural variations (SVs) are common in cancer. Although a small fraction of SVs in breast and ovarian cancers can be attributed to homologous recombination deficiency, the underlying molecular mechanisms for the vast majority of somatic SVs remain unclear. Here, we focus on the roles of transcription and DNA replication collisions in genomic instability in cancer. Such collisions are unavoidable in cells since both transcription and replication use the same DNA as template. We hypothesized that transcription replication collisions (TRCs), if not properly repaired, would lead to collapsed replication forks and result in SVs. To this end, we studied somatic SVs in 5994 high-coverage whole-genome sequenced primary and metastatic tumors from three independent pan-cancer cohorts. A total of 12 conserved SV signatures, representing independent molecular mechanisms, were deconvoluted from these cohorts using non-negative matrix factorization approach. We detected replicated-strand bias, the expected footprint of transcription-replication collision, in large tandem duplications (TDs) across multiple cohorts. This bias was only observed in expressed genes, consistent with TRCs depending on transcription activity. Large TDs were abundant in female-specific (breast, ovarian and uterus), upper gastric-intestinal tract and prostate cancers. They were associated with worse patient survival and TP53 and CDK12 mutations. CDK12 is a cyclin-dependent kinase (CDK) and a key regulator of transcription elongation. Deleting or suppressing CDK12 using CRISPR-Cas9 in prostate cancer cell lines not only increased RNA:DNA hybrids (R-loops), but also promoted TRCs, suggesting a mechanism by which dysregulation of a transcriptional CDK may lead to genomic instability. Finally, using existing large-scale drug screening data, we found that cancer cell lines with abundant large TDs were significantly more sensitive to the WEE1 inhibitor, MK-1775, which we experimentally validated in prostate cancer cells lacking CDK12. In summary, our data suggest that large TDs in cancer form due to impaired TRC repair and can be used as a biomarker for prognosis and treatment. Citation Format: Yang Yang, Michelle Badura, Patrick O’Leary, Emily Egusa, Troy Robinson, Xiaoming Zhong, Jason Swinderman, Minkyu Kim, Haolong Li, Alan Ashworth, Felix Feng, Jonathan Chou, Lixing Yang. Large tandem duplications in cancer resulting from transcription and DNA replication collisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 299.

Multi-omics investigation reveals functional specialization of transcriptional cyclin dependent kinases in cancer biology

Transcriptional addiction is recognized as a valid therapeutic target in cancer, whereby the dependency of cancer cells on oncogenic transcriptional regulators may be pharmacologically exploited. However, a comprehensive understanding of the key factors within the transcriptional machinery that might afford a useful therapeutic window remains elusive. Herein, we present a cross-omics investigation into the functional specialization of the transcriptional cyclin dependent kinases (tCDKs) through analysis of high-content genetic dependency, gene expression, patient survival, and drug response datasets. This analysis revealed specialization among tCDKs in terms of contributions to cancer cell fitness, clinical prognosis, and interaction with oncogenic signaling pathways. CDK7 and CDK9 stand out as the most relevant targets, albeit through distinct mechanisms of oncogenicity and context-dependent contributions to cancer survival and drug sensitivity. Genetic ablation of CDK9, but not CDK7, mimics the effect on cell viability the loss of key components of the transcriptional machinery. Pathway analysis of genetic co-dependency and drug sensitivity data show CDK7 and CDK9 have distinct relationships with major oncogenic signatures, including MYC and E2F targets, oxidative phosphorylation, and the unfolded protein response. Altogether, these results inform the improved design of therapeutic strategies targeting tCDKs in cancer.

Promising Anticancer Activity of Multitarget Cyclin Dependent Kinase Inhibitors against Human Colorectal Carcinoma Cells.

Colorectal cancer (CRC) is the third leading cause of cancer death worldwide, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Yet, efficacy of multitarget CDK inhibitors as therapeutic agents has not been much explored against CRC. The anticancer potential of multitarget CDK inhibitor riviciclib (also known as P276-00), was investigated against CRC cell lines of varied genetic background. Cytotoxicity of riviciclib - potent CDK1, CDK4 and CDK9-specific inhibitor was evaluated in vitro. Further, its effect on clonogenic potential, cell cycle, apoptosis and transcription was tested using colony forming assay, flow cytometry and western blot analysis, respectively. Also, efficacy of riviciclib in combination with standard chemotherapeutic agents was assessed. Dependency of CRC cells on specific CDKs for their survival was confirmed using siRNA studies. Riviciclib exerted significant cytotoxicity against CRC cells and inhibited their colony forming potential. It induced apoptosis along with inhibition of cell cycle CDKs and cyclins as well as transcriptional CDKs and cyclins. Moreover, dual combination of riviciclib with standard chemotherapeutic drugs exhibited synergism in CRC cells. siRNA studies indicated that CRC cells are dependent on specific CDKs for their survival which are targets of riviciclib. This study provides evidence that multitarget CDK inhibitors can serve as promising therapeutic agents against CRC alone or in combination.

Abstract B075: Sirtuin 6 histone deacetylase is required for the integrated stress response and resistance to inhibition of transcriptional cyclin dependent kinases in Pancreatic Ductal Adenocarcinoma

Abstract Pancreatic Ductal Adenocarcinoma (PDA) can be characterized by two distinct transcriptional subtypes: Classical and Basal-like. The basal PDA subtype is more aggressive and has the worst overall survival. Previous work has shown that Sirtuin 6 histone deacetylase (SIRT6) acts as a tumor suppressor and cooperates with oncogenic KRAS in GEMM models of PDA. Our study identifies SIRT6 as a biomarker for basal PDA and investigates the underlying sensitivity of basal PDA to inhibition of transcriptional cyclin dependent kinases (CDKs). Through analysis of data from hundreds of human PDA tumors, we identified that low SIRT6 expression correlates with basal PDA while the converse is true for classical PDA. Using many in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts of PDA, we also discovered that basal PDA is uniquely sensitive to the covalent CDK7/12/13 inhibitor THZ1 as well as other inhibitors of transcriptional CDKs. Importantly, removal of SIRT6 can sensitize SIRT6 high/classical PDA to THZ1. Further investigation identified that SIRT6 low/basal PDA has an inactivated Integrated Stress Response (ISR), which is primarily driven by ATF4. To determine how loss of SIRT6 leads to an inability to activate the ISR, we have integrated use of human PDA cell lines, and organoids as well as murine PDA GEMMs where SIRT6 has been deleted or downregulated. We uncovered that SIRT6 regulates the ISR through control of ATF4 translation, specifically through a 3’ UTR open reading frame (uORF) dependent mechanism. To determine whether ISR activation could protect basal PDA cells from transcriptional CDK inhibition, we manipulated expression of ISR pathway components. We found that activation of the ISR in SIRT6 low/basal PDA reduces sensitivity to THZ1, while inactivation of the ISR sensitizes SIRT6 high/classical PDA to this inhibitor. Interestingly, both basal and classical PDA exhibit increased phosphorylation of eIF2a upon inhibition of transcriptional CDKs, which typically indicates activation of the ISR in response to stress. However, this activation of the ISR remains incomplete since ATF4 translation is not upregulated in basal PDA in the absence of SIRT6. Therefore, we discovered that loss of SIRT6 sensitizes basal PDA to inhibition of transcriptional CDKs through an inability to launch a functional ISR response. Furthermore, we have uncovered an important biomarker which controls a stress-induced transcriptional program that may be exploited with targeted therapies in a particularly aggressive PDA subtype. Citation Format: Jessica Gianopulos, Nithya Kartha, Zachary Schrank, Stephanie Dobersch, Sarah Cavender, Bryan Kynnap, Adrianne Wallace-Povirk, Cynthia Wladyka, Juan Santana, Jaeseung C. Kim, Angela Yu, Caroline Bridgwater, Kathrin Fuchs, Sarah Dysinger, Aaron Lampano, Faiyaz Notta, David Price, Andrew Hsieh, Sunil Hingorani, Sita Kugel. Sirtuin 6 histone deacetylase is required for the integrated stress response and resistance to inhibition of transcriptional cyclin dependent kinases in Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B075.

Design and Synthesis of a 2-Amino-pyridine Derivative as a Potent CDK8 Inhibitor for Anti-colorectal Cancer Therapy.

CDK8 is a transcriptional cyclin-dependent kinase and considered as a potential target in colon cancer therapeutics. Here, a novel selective CDK8 inhibitor was identified against colon cancer in vivo. Specifically, based on the structural information of the sorafenib-bound CDK8 structure, a series of novel 2-amino-pyridine derivatives were designed, synthesized, and evaluated. Among them, compound 29 showed strong inhibitory activity against CDK8 with an IC50 value of 46 nM and favorable selectivity. And there is an apparent interaction between the endogenous or overexpressed CDK8 and biotinylated-29. This compound exhibited antiproliferation potency on colon cancer cell lines with a high CDK8 expression level, suppressed the activation of WNT/β-catenin and transcriptional activity of the TCF family, and induced G1 phase arrested in HCT-116 cells. In addition, this compound showed potent activity against sorafenib-resistant HCT-116 cells. What's more, it exhibited low toxicity and suitable pharmacokinetic (PK) profiles and showed preferable antitumor effects in vivo.

Targeting the Heterogeneous Genomic Landscape in Triple-Negative Breast Cancer through Inhibitors of the Transcriptional Machinery.

Simple SummaryTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with limited options for therapy. Cancer development and maintenance is dependent on the production of oncogenic proteins. In TNBC, the production of many of these proteins is changed, and therapeutic targeting of one of these proteins is often not effective. However, a common step in the production of these proteins, transcription, can be effectively targeted through inhibition of the transcriptional machinery. These inhibitors can specifically suppress the production of oncogenic proteins important for TNBC. At the same time, the altered production of these proteins might interfere with the sensitivity of these cancer cells to these inhibitors. This review provides an overview of the effects of inhibitors of the transcriptional machinery on the abnormal oncogenic protein production in TNBC. This overview thereby highlights the research further needed in this field, and the potential opportunities of using these inhibitors for TNBC.Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer defined by lack of the estrogen, progesterone and human epidermal growth factor receptor 2. Although TNBC tumors contain a wide variety of oncogenic mutations and copy number alterations, the direct targeting of these alterations has failed to substantially improve therapeutic efficacy. This efficacy is strongly limited by interpatient and intratumor heterogeneity, and thereby a lack in uniformity of targetable drivers. Most of these genetic abnormalities eventually drive specific transcriptional programs, which may be a general underlying vulnerability. Currently, there are multiple selective inhibitors, which target the transcriptional machinery through transcriptional cyclin-dependent kinases (CDKs) 7, 8, 9, 12 and 13 and bromodomain extra-terminal motif (BET) proteins, including BRD4. In this review, we discuss how inhibitors of the transcriptional machinery can effectively target genetic abnormalities in TNBC, and how these abnormalities can influence sensitivity to these inhibitors. These inhibitors target the genomic landscape in TNBC by specifically suppressing MYC-driven transcription, inducing further DNA damage, improving anti-cancer immunity, and preventing drug resistance against MAPK and PI3K-targeted therapies. Because the transcriptional machinery enables transcription and propagation of multiple cancer drivers, it may be a promising target for (combination) treatment, especially of heterogeneous malignancies, including TNBC.

P10.12.A CDK12/CDK13 inhibition disrupts a transcriptional program critical for glioblastoma survival

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&amp;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.

Synthesis of 4-styrylpyrazoles and Evaluation of their Inhibitory Effects on Cyclin-dependent Kinases.

Cycle-regulating and transcriptional cyclin-dependent kinases (CDKs) are attractive targets in cancer drug development. Several CDK inhibitors have already been obtained or are close to regulatory approval for clinical applications. Phenylazopyrazole CAN508 has been described as the first selective CDK9 inhibitor with an IC50 of 350 nM. Since the azo-moiety is not a suitable functionality for drugs due to pharmacological reasons, the preparation of carbo-analogues of CAN508 with similar biological activities is desirable. The present work is focused on the synthesis of carbo-analogues similar to CAN508 and their CDK inhibition activity. Herein, the synthesis of 21 novel carbo analogues of CAN508 and their intermediates is reported. Subsequently, target compounds 8a - 8u were evaluated for protein kinase inhibition (CDK2/cyclin E, CDK4/cyclin D, CDK9/cyclin T) and antiproliferative activities in cell lines (K562, MCF-7, MV4-11). Moreover, the binding mode of derivative 8s in the active site of CDK9 was modelled. Compounds 8a - 8u were obtained from key intermediate 7, which was prepared by linear synthesis involving Vilsmeier-Haack, Knoevenagel, Hunsdiecker, and Suzuki-Miyaura reactions. Styrylpyrazoles 8t and 8u were the most potent CDK9 inhibitors with IC50 values of approximately 1 μM. Molecular modelling suggested binding in the active site of CDK9. The flow cytometric analysis of MV4-11 cells treated with the most active styrylpyrazoles showed a significant G1-arrest. The prepared styrylpyrazoles showed inhibition activity towards CDKs and can provide a novel chemotype of kinase inhibitors.

Transcriptional CDK Inhibitors as Potential Treatment Option for Testicular Germ Cell Tumors.

Simple SummaryType II testicular germ cell tumors are a severe type of cancer in young men demanding alternative treatment options to conventional chemotherapy with less side effects. In particular, patients with chemotherapy-resistant tumors face a bad prognosis and low survival rates. In other tumor entities, transcriptional cyclin-dependent kinases (7/8/9/12/13) have been demonstrated to be effective targets. Here, we studied the effects of transcriptional cyclin-dependent kinase inhibitors on a cellular and molecular level. We found several inhibitors to be highly cytotoxic for certain testicular germ cell tumor cell lines while leaving a somatic (fibroblast) control cell line unaffected. This opens up a novel field for effective and specified treatment of type II testicular germ cell tumors.Type II testicular germ cell tumors (TGCT) are the most frequently diagnosed solid malignancy in young men. Up to 15% of patients with metastatic non-seminomas show cisplatin resistance and a very poor survival rate due to lacking treatment options. Transcriptional cyclin-dependent kinases (CDK) have been shown to be effective targets in the treatment of different types of cancer. Here, we investigated the effects of the CDK inhibitors dinaciclib, flavopiridol, YKL-5-124, THZ1, NVP2, SY0351 and THZ531. An XTT viability assay revealed a strong cytotoxic impact of CDK7/12/13 inhibitor SY0351 and CDK9 inhibitor NVP2 on the TGCT wild-type cell lines (2102EP, NCCIT, TCam2) and the cisplatin-resistant cell lines (2102EP-R, NCCIT-R). The CDK7 inhibitor YKL-5-124 showed a strong impact on 2102EP, 2102EP-R, NCCIT and NCCIT-R cell lines, leaving the MPAF control cell line mostly unaffected. FACS-based analysis revealed mild effects on the cell cycle of 2102EP and TCam2 cells after SY0351, YKL-5-124 or NVP2 treatment. Molecular analysis showed a cell-line-specific response for SY0351 and NVP2 inhibition while YKL-5-124 induced similar molecular changes in 2102EP, TCam2 and MPAF cells. Thus, after TGCT subtype determination, CDK inhibitors might be a potential alternative for optimized and individualized therapy independent of chemotherapy sensitivity.

Preclinical Modeling of Leiomyosarcoma Identifies Susceptibility to Transcriptional CDK Inhibitors through Antagonism of E2F-Driven Oncogenic Gene Expression.

Leiomyosarcoma (LMS) is a neoplasm characterized by smooth muscle differentiation, complex copy-number alterations, tumor suppressor loss, and the absence of recurrent driver mutations. Clinical management for advanced disease relies on the use of empiric cytotoxic chemotherapy with limited activity, and novel targeted therapies supported by preclinical research on LMS biology are urgently needed. A lack of fidelity of established LMS cell lines to their mesenchymal neoplasm of origin has limited translational understanding of this disease, and few other preclinical models have been established. Here, we characterize patient-derived xenograft (PDX) models of LMS, assessing fidelity to their tumors of origin and performing preclinical evaluation of candidate therapies. We implanted 49 LMS surgical samples into immunocompromised mice. Engrafting tumors were characterized by histology, targeted next-generation sequencing, RNA sequencing, and ultra-low passage whole-genome sequencing. Candidate therapies were selected based on prior evidence of pathway activation or high-throughput dynamic BH3 profiling. We show that LMS PDX maintain the histologic appearance, copy-number alterations, and transcriptional program of their parental tumors across multiple xenograft passages. Transcriptionally, LMS PDX cocluster with paired LMS patient-derived samples and differ primarily in host-related immunologic and microenvironment signatures. We identify susceptibility of LMS PDX to transcriptional cyclin-dependent kinase (CDK) inhibition, which disrupts an E2F-driven oncogenic transcriptional program and inhibits tumor growth. Our results establish LMS PDX as valuable preclinical models and identify strategies to discover novel vulnerabilities in this disease. These data support the clinical assessment of transcriptional CDK inhibitors as a therapeutic strategy for patients with LMS.