Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

CDK7 kinase activity promotes RNA polymerase II promoter escape by facilitating initiation factor release.

Cyclin-dependent kinase 7 (CDK7), part of the general transcription factor TFIIH, promotes gene transcription by phosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II). Here, we combine rapid CDK7 kinase inhibition with multi-omics analysis to unravel the direct functions of CDK7 in human cells. CDK7 inhibition causes RNA Pol II retention at promoters, leading to decreased RNA Pol II initiation and immediate global downregulation of transcript synthesis. Elongation, termination, and recruitment of co-transcriptional factors are not directly affected. Although RNA Pol II, initiation factors, and Mediator accumulate at promoters, RNA Pol II complexes can also proceed into gene bodies without promoter-proximal pausing while retaining initiation factors and Mediator. Further downstream, RNA Pol II phosphorylation increases and initiation factors and Mediator are released, allowing recruitment of elongation factors and an increase in RNA Pol II elongation velocity. Collectively, CDK7 kinase activity promotes the release of initiation factors and Mediator from RNA Pol II, facilitating RNA Pol II escape from the promoter.

Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition.

Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.

CDK7 regulates organ size and tumor growth by safeguarding the Hippo pathway effector Yki/Yap/Taz in the nucleus.

Hippo signaling controls organ size and tumor progression through a conserved pathway leading to nuclear translocation of the transcriptional effector Yki/Yap/Taz. Most of our understanding of Hippo signaling pertains to its cytoplasmic regulation, but how the pathway is controlled in the nucleus remains poorly understood. Here we uncover an evolutionarily conserved mechanism by which CDK7 promotes Yki/Yap/Taz stabilization in the nucleus to sustain Hippo pathway outputs. We found that a modular E3 ubiquitin ligase complex CRL4DCAF12 binds and targets Yki/Yap/Taz for ubiquitination and degradation, whereas CDK7 phosphorylates Yki/Yap/Taz at S169/S128/S90 to inhibit CRL4DCAF12 recruitment, leading to Yki/Yap/Taz stabilization. As a consequence, inactivation of CDK7 reduced organ size and inhibited tumor growth, which could be reversed by restoring Yki/Yap activity. Our study identifies an unanticipated layer of Hippo pathway regulation, defines a novel mechanism by which CDK7 regulates tissue growth, and implies CDK7 as a drug target for Yap/Taz-driven cancer.

Selective inhibition of CDK7 reveals high-confidence targets and new models for TFIIH function in transcription.

CDK7 associates with the 10-subunit TFIIH complex and regulates transcription by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII). Few additional CDK7 substrates are known. Here, using the covalent inhibitor SY-351 and quantitative phosphoproteomics, we identified CDK7 kinase substrates in human cells. Among hundreds of high-confidence targets, the vast majority are unique to CDK7 (i.e., distinct from other transcription-associated kinases), with a subset that suggest novel cellular functions. Transcription-associated factors were predominant CDK7 substrates, including SF3B1, U2AF2, and other splicing components. Accordingly, widespread and diverse splicing defects, such as alternative exon inclusion and intron retention, were characterized in CDK7-inhibited cells. Combined with biochemical assays, we establish that CDK7 directly activates other transcription-associated kinases CDK9, CDK12, and CDK13, invoking a "master regulator" role in transcription. We further demonstrate that TFIIH restricts CDK7 kinase function to the RNAPII CTD, whereas other substrates (e.g., SPT5 and SF3B1) are phosphorylated by the three-subunit CDK-activating kinase (CAK; CCNH, MAT1, and CDK7). These results suggest new models for CDK7 function in transcription and implicate CAK dissociation from TFIIH as essential for kinase activation. This straightforward regulatory strategy ensures CDK7 activation is spatially and temporally linked to transcription, and may apply toward other transcription-associated kinases.

In vitro analysis of RNA polymerase II elongation complex dynamics.

RNA polymerase II elongation complexes (ECs) were assembled from nuclear extract on immobilized DNA templates and analyzed by quantitative mass spectrometry. Time-course experiments showed that initiation factor TFIIF can remain bound to early ECs, while levels of core elongation factors Spt4-Spt5, Paf1C, Spt6-Spn1, and Elf1 remain steady. Importantly, the dynamic phosphorylation patterns of the Rpb1 C-terminal domain (CTD) and the factors that recognize them change as a function of postinitiation time rather than distance elongated. Chemical inhibition of Kin28/Cdk7 in vitro blocks both Ser5 and Ser2 phosphorylation, affects initiation site choice, and inhibits elongation efficiency. EC components dependent on CTD phosphorylation include capping enzyme, cap-binding complex, Set2, and the polymerase-associated factor (PAF1) complex. By recapitulating many known features of in vivo elongation, this system reveals new details that clarify how EC-associated factors change at each step of transcription.

Disruption of CDK7 signaling leads to catastrophic chromosomal instability coupled with a loss of condensin-mediated chromatin compaction.

Chromatin organization is highly dynamic and modulates DNA replication, transcription, and chromosome segregation. Condensin is essential for chromosome assembly during mitosis and meiosis, as well as maintenance of chromosome structure during interphase. While it is well established that sustained condensin expression is necessary to ensure chromosome stability, the mechanisms that control its expression are not yet known. Herein, we report that disruption of cyclin-dependent kinase 7 (CDK7), the core catalytic subunit of CDK-activating kinase, leads to reduced transcription of several condensin subunits, including structural maintenance of chromosomes 2 (SMC2). Live and static microscopy revealed that inhibiting CDK7 signaling prolongs mitosis and induces chromatin bridge formation, DNA double-strand breaks, and abnormal nuclear features, all of which are indicative of mitotic catastrophe and chromosome instability. Affirming the importance of condensin regulation by CDK7, genetic suppression of the expression of SMC2, a core subunit of this complex, phenocopies CDK7 inhibition. Moreover, analysis of genome-wide chromatin conformation using Hi-C revealed that sustained activity of CDK7 is necessary to maintain chromatin sublooping, a function that is ascribed to condensin. Notably, the regulation of condensin subunit gene expression is independent of superenhancers. Together, these studies reveal a new role for CDK7 in sustaining chromatin configuration by ensuring the expression of condensin genes, including SMC2.

Cimicifugin, a broad-spectrum inhibitor of Theileria annulata and Plasmodium falciparum CDK7.

Cyclin-dependent kinase 7 is an attractive therapeutic target for the treatment of cancers, and a previous report suggested that Plasmodium falciparum CDK7 is a potential drug target for developing new anti-malarial drugs. In this study, we aimed to characterize and evaluate the drug target potential of Theileria annulata CDK7. Theileria annulata is responsible for tropical theileriosis, which induces a phenotype similar to cancerous cells like immortalization, hyperproliferation, and dissemination. Virtual screening of the MyriaScreen II library predicted 14 compounds with high binding energies to the ATP-binding pocket of TaCDK7. Three compounds (cimicifugin, ST092793, and ST026925) of these 14 compounds were non-cytotoxic to the uninfected bovine cells (BoMac cells). Cimicifugin treatment led to the activation of the extrinsic apoptosis pathway and induced autophagy in T. annulata-infected cells. Furthermore, cimicifugin also inhibited the growth of P. falciparum, indicating that it has both anti-theilerial and anti-malarial activities and that TaCDK7 and PfCDK7 are promising drug targets.

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.

CDK7 in breast cancer: mechanisms of action and therapeutic potential.

Cyclin-dependent kinase 7 (CDK7) serves as a pivotal regulator in orchestrating cellular cycle dynamics and gene transcriptional activity. Elevated expression levels of CDK7 have been ubiquitously documented across a spectrum of malignancies and have been concomitantly correlated with adverse clinical outcomes. This review delineates the biological roles of CDK7 and explicates the molecular pathways through which CDK7 exacerbates the oncogenic progression of breast cancer. Furthermore, we synthesize the extant literature to provide a comprehensive overview of the advancement of CDK7-specific small-molecule inhibitors, encapsulating both preclinical and clinical findings in breast cancer contexts. The accumulated evidence substantiates the conceptualization of CDK7 as a propitious therapeutic target in breast cancer management.

Discovery of novel co-degradation CK1α and CDK7/9 PROTACs with p53 activation for treating acute myeloid leukemia.

Reactivating p53 activity to restore its anticancer function is an attractive cancer treatment strategy. In this study, we designed and synthesized a series of novel PROTACs to reactivate p53 via the co-degradation of CK1α and CDK7/9 proteins. Bioactivity studies showed that the selected PROTAC 13i exhibited potency antiproliferative activity in MV4-11 (IC50 = 0.096 ± 0.012 µM) and MOLM-13 (IC50 = 0.072 ± 0.014 µM) cells, and induced apoptosis of MV4-11 cells. Western-blot analysis showed that PROTAC 13i triple CK1α and CDK7/9 protein degradation resulted in the significantly increased expression of p53. At the same time, the transcriptional repression due to the degradation significantly reduced downstream gene expression of MYC, MDM2, BCL-2 and MCL-1, and reduced the inflammatory cytokine levels of TNF-α, IL-1ß and IL-6 in PMBCs. These results indicate the beneficial impact of simultaneous CK1α and CDK7/9 degradation for acute myeloid leukemia therapy.

CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma.

Melanoma cell phenotype switching between differentiated melanocytic and undifferentiated mesenchymal-like states drives metastasis and drug resistance. CDK7 is the serine/threonine kinase of the basal transcription factor TFIIH. We show that dedifferentiation of melanocytic-type melanoma cells into mesenchymal-like cells and acquisition of tolerance to targeted therapies is achieved through chronic inhibition of CDK7. In addition to emergence of a mesenchymal-type signature, we identify a GATA6-dependent gene expression program comprising genes such as AMIGO2 or ABCG2 involved in melanoma survival or targeted drug tolerance, respectively. Mechanistically, we show that CDK7 drives expression of the melanocyte lineage transcription factor MITF that in turn binds to an intronic region of GATA6 to repress its expression in melanocytic-type cells. We show that GATA6 expression is activated in MITF-low melanoma cells of patient-derived xenografts. Taken together, our data show how the poorly characterized repressive function of MITF in melanoma participates in a molecular cascade regulating activation of a transcriptional program involved in survival and drug resistance in melanoma.

Discovery of SHR5428 as a selective and noncovalent inhibitor of CDK7.

Cyclin dependent kinase 7 (CDK7) is an attractive target in tumor indications via regulating both cell cycle and transcription. Here, SHR5428 was discovered as a selective and noncovalent CDK7 inhibitor with highly potent CDK7 enzymatic activity and triple negative breast cancer cellular activity on MDA-MB-468 cell. SHR5428 also displayed favorable pharmacokinetic properties in different preclinical species such as mouse, rat and dog, and showed high selectivity over CDK1, CDK2, CDK4, CDK6, CDK9, CDK12 in CDK family. Furthermore, the computational modeling has shed some light on this mechanism. Additionally the in vivo efficacy study in a breast cancer cell line (HCC70 cell) derived xenograft mouse model proved SHR5428 to be orally efficacious with dose-dependent tumor growth inhibition.

The Interactive Complex between Cytomegalovirus Kinase vCDK/pUL97 and Host Factors CDK7-Cyclin H Determines Individual Patterns of Transcription in Infected Cells.

The infection of human cytomegalovirus (HCMV) is strongly determined by the host-cell interaction in a way that the efficiency of HCMV lytic replication is dependent on the regulatory interplay between viral and cellular proteins. In particular, the activities of protein kinases, such as cyclin-dependent kinases (CDKs) and the viral CDK ortholog (vCDK/pUL97), play an important role in both viral reproduction and virus-host interaction. Very recently, we reported on the complexes formed between vCDK/pUL97, human cyclin H, and CDK7. Major hallmarks of this interplay are the interaction between cyclin H and vCDK/pUL97, which is consistently detectable across various conditions and host cell types of infection, the decrease or increase in pUL97 kinase activity resulting from cyclin H knock-down or elevated levels, respectively, and significant trans-stimulation of human CDK7 activity by pUL97 in vitro. Due to the fact that even a ternary complex of vCDK/pUL97-cyclin H-CDK7 can be detected by coimmunoprecipitation and visualized by bioinformatic structural modeling, we postulated a putative impact of the respective kinase activities on the patterns of transcription in HCMV-infected cells. Here, we undertook a first vCDK/pUL97-specific transcriptomic analysis, which combined conditions of fully lytic HCMV replication with those under specific vCDK/pUL97 or CDK7 drug-mediated inhibition or transient cyclin H knockout. The novel results were further strengthened using bioinformatic modeling of the involved multi-protein complexes. Our data underline the importance of these kinase activities for the C-terminal domain (CTD) phosphorylation-driven activation of host RNA polymerase in HCMV-infected cells. The impact of the individual experimental conditions on differentially expressed gene profiles is described in detail and discussed.

1,6-Hexanediol, commonly used to dissolve liquid-liquid phase separated condensates, directly impairs kinase and phosphatase activities.

The concept of liquid-liquid phase separation (LLPS) has emerged as an intriguing mechanism for the organization of membraneless compartments in cells. The alcohol 1,6-hexanediol is widely used as a control to dissolve LLPS assemblies in phase separation studies in diverse fields. However, little is known about potential side effects of 1,6-hexanediol, which could compromise data interpretation and mislead the scientific debate. To examine this issue, we analyzed the effect of 1,6-hexanediol on the activities of various enzymes in vitro. Already at 1% volume concentration, 1,6-hexanediol strongly impaired kinases and phosphatases and partly blocked DNA polymerases, while it had no effect on DNase activity. At concentrations that are usually used to dissolve LLPS droplets (5-10%), both kinases and phosphatases were virtually inactive. Given the widespread function of protein phosphorylation in cells, our data argue for a careful review of 1,6-hexanediol in phase separation studies.

TGF-ß/activin signaling promotes CDK7 inhibitor resistance in triple-negative breast cancer cells through upregulation of multidrug transporters.

Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-ß) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-ß family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-ß/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-ß/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors.

Elevation of effective p53 expression sensitizes wild-type p53 breast cancer cells to CDK7 inhibitor THZ1.

BACKGROUND: The cyclin-dependent kinase 7 (CDK7) inhibitor THZ1 represses multiple cancer cells. However, its tumor-repressive efficiency in wild-type p53 breast cancer cells remains controversial. METHODS: We conducted various assays, including CCK8, colony formation, flow cytometry, western blotting, and lactate dehydrogenase release detection, to clarify whether p53 elevation sensitizes breast cancer cells to THZ1. RESULTS: We found that upregulating functional p53 contributes to the increased sensitivity of breast cancer cells to THZ1. Increased THZ1 sensitivity requires active p53 and an intact p53 pathway, which was confirmed by introducing exogenous wild-type p53 and the subsequent elevation of THZ1-mediated tumor suppression in breast cancer cells carrying mutant p53. We confirmed that p53 accumulates in the nucleus and mitochondria during cell death. Furthermore, we identified extensive transcriptional disruption, rather than solely CDK7 inhibition, as the mechanism underlying the nutlin-3 and THZ1-induced death of breast cancer cells. Finally, we observed the combined nutlin-3 and THZ1 treatment amplified gasdermin E cleavage. CONCLUSION: Enhanced sensitivity of breast cancer cells to THZ1 can be achieved by increasing effective p53 expression. Our approach may serve as a potential treatment for patients with breast cancer resistant to regular therapies. Video Abstract.

THZ1 targeting CDK7 suppresses c-KIT transcriptional activity in gastrointestinal stromal tumours.

BACKGROUND: Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the gastrointestinal tract and are characterized by activating mutations of c-KIT or PDGFRa receptor tyrosine kinases (RTKs). Despite the clinical success of tyrosine kinase inhibitors (TKIs), more than half of GIST patients develop resistance due to a second mutation. Cyclin-dependent kinase 7 (CDK7) is the catalytic subunit of CDK-activating kinase (CAK), and it plays an important role in the regulation of cell cycle transitions and gene transcription. THZ1, a CDK7 inhibitor, exhibits a dose-dependent inhibitory effect in various cancers. METHODS: Data from the public GEO database and tissue microarray were used to analyse the gene expression levels of CDKs in GISTs. The impact of CDK7 knockdown and the CDK7 inhibitor THZ1 on GIST progression was investigated in vitro using CCK-8, colony formation, and flow cytometry assays and in vivo using a xenograft mouse model. RNA sequencing was performed to investigate the mechanism of GIST cell viability impairment mediated by THZ1 treatment. RESULTS: Our study demonstrated that CDK7 is relatively overexpressed in high-risk GISTs and predicts a poor outcome. A low concentration of THZ1 exhibited a pronounced antineoplastic effect in GIST cells in vivo and in vitro. Moreover, THZ1 exerted synergistic anticancer effects with imatinib. THZ1 treatment resulted in transcriptional modulation by inhibiting the phosphorylation of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII). c-KIT, an oncogene driver of GIST, was transcriptionally repressed by THZ1 treatment or CDK7 knockdown. Transcriptome sequencing analysis showed that OSR1 acts as a downstream target of CDK7 and regulates c-KIT expression. Taken together, our results highlight elevated CDK7 expression as a predictor of poor outcome in GIST and present the combination of CDK7 and RTK inhibitors as a potent therapeutic strategy to improve the efficacy of GIST treatment. Video abstract.

CDK7 inhibition suppresses aberrant hedgehog pathway and overcomes resistance to smoothened antagonists.

The aberrant hedgehog (Hh) pathway plays important roles in multiple cancer types, therefore serving as a promising drug target. Current clinically available hedgehog-targeted drugs act mostly by antagonizing the upstream component smoothened; however, both primary and acquired resistance to FDA-approved smoothened inhibitor (SMOi) drugs have been described. We have recently demonstrated that the BET inhibitor effectively suppresses SMOi-resistant Hh-driven cancers through antagonizing transcription of GLI1 and GLI2, the core transcriptional factors of Hh pathway, suggesting epigenetic or transcriptional targeted therapy represents an anti-Hh therapeutic strategy that can overcome SMOi resistance. Here we performed an unbiased screening of epigenetic or transcriptional targeted small molecules to test their inhibitory effects on GLI1 and GLI2 transcription or cell viability of Hh-driven tumor lines. THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), is identified as the top hit in our screening. We then confirmed that antagonizing CDK7 by either small-molecule inhibitors or the CRISPR-Cas9 approach causes substantial suppression of GLI1 and GLI2 transcription, resulting in effective inhibition of Hh-driven cancers in vitro and in vivo. More importantly, antagonizing CDK7 retains inhibitory activity against Hh-driven cancers with almost all so-far described primary or acquired SMOi resistance. Furthermore, we reveal a synergy between CDK7 inhibition and BET inhibition on antagonizing aberrant Hh pathway and Hh-driven cancers that are either responsive or resistant to SMOi. Our results illustrate transcriptional inhibition through targeting CDK7 as a promising therapeutic strategy for treating Hh-driven cancers, especially those with primary or acquired resistance to SMOi drugs.

Control of Expression of Key Cell Cycle Enzymes Drives Cell Line-Specific Functions of CDK7 in Human PDAC Cells.

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.