Mediator Kinase Research Articles

Mediator kinase inhibitors suppress triple-negative breast cancer growth and extend tumor suppression by mTOR and AKT inhibitors

Triple-negative breast cancers (TNBC) are treated primarily by chemotherapy and lack clinically validated therapeutic targets. In particular, inhibitors of the PI3K/AKT/mTOR pathway, abnormally activated in many breast cancers, failed to achieve clinical efficacy in TNBC due to the development of adaptive drug resistance, which is largely driven by the transcriptomic plasticity of TNBC. Expression of CDK8/19 Mediator kinases that control transcriptional reprogramming correlates with relapse-free survival and treatment failure in breast cancer patients, including TNBC. We now investigated how CDK8/19 inhibitors affect the growth of TNBC tumors and their response to mTOR and AKT inhibitors. In contrast to the effects of most anticancer drugs, all the tested human TNBC models (including patient-derived xenografts) responded to CDK8/19 inhibitors in vivo even when they did not respond in vitro. Furthermore, CDK8/19 inhibition extended the host survival of established lung metastases in a murine TNBC model, where the primary tumors were not significantly affected. CDK8/19 inhibitors synergized with an mTORC1 inhibitor everolimus and a pan-AKT inhibitor capivasertib in vitro and strongly potentiated these drugs in long-term in vivo studies. Transcriptomic analysis of tumors that responded or became adapted to everolimus revealed that drug adaptation in vivo was associated with major transcriptional changes in both tumor and stromal cells. Combining everolimus with a CDK8/19 inhibitor counteracted many of these changes and induced combination-specific effects on the expression of multiple genes that affect tumor growth. These results warrant the exploration of CDK8/19 Mediator kinase inhibitors as a new type of drugs for TNBC therapy.

CDK8 of the mediator kinase module connects leaf development to the establishment of correct stomata patterning by regulating the levels of the transcription factor SPEECHLESS (SPCH).

The components of the mediator kinase module are highly conserved across all eukaryotic lineages, and cyclin-dependent kinase 8 (CDK8) is essential for correct cell proliferation and differentiation in diverse eukaryotic systems. We show that CDK8 couples leaf development with the establishment of correct stomata patterning for prevailing CO2 conditions. In Arabidopsis, the basic helix-loop-helix(bHLH) transcription factor SPEECHLESS(SPCH) controls cellular entry into the stomatal cell lineage, and CDK8 interacts with and phosphorylates SPCH, controlling SPCH protein levels and thereby also expression of the SPCH target genes encoding key regulators of cell fate and asymmetric cell divisions. The lack of the CDK8-mediated control of SPCH results in an increased number of meristemoid and guard mother cells, and increased stomata index in the cdk8 mutants. Increasing atmospheric CO2 concentrations trigger a developmental programme controlling cell entry into stomatal lineage by limiting the asymmetric divisions. In cdk8, the number of meristemoids and guard mother cells remains the same under ambient and high CO2 concentrations, as the accumulated levels of SPCH caused by the lack of CDK8 appear to override the negative regulation of increased CO2. Thus, our work provides novel mechanistic understanding of how plants alter critical leaf properties in response to increasing atmospheric CO2.

Mediator kinase inhibition impedes transcriptional plasticity and prevents resistance to ERK/MAPK-targeted therapy in KRAS-mutant cancers

Acquired resistance remains a major challenge for therapies targeting oncogene activated pathways. KRAS is the most frequently mutated oncogene in human cancers, yet strategies targeting its downstream signaling kinases have failed to produce durable treatment responses. Here, we developed multiple models of acquired resistance to dual-mechanism ERK/MAPK inhibitors across KRAS-mutant pancreatic, colorectal, and lung cancers, and then probed the long-term events enabling survival against this class of drugs. These studies revealed that resistance emerges secondary to large-scale transcriptional adaptations that are diverse and cell line-specific. Transcriptional reprogramming extends beyond the well-established early response, and instead represents a dynamic, evolved process that is refined to attain a stably resistant phenotype. Mechanistic and translational studies reveal that resistance to dual-mechanism ERK/MAPK inhibition is broadly susceptible to manipulation of the epigenetic machinery, and that Mediator kinase, in particular, can be co-targeted at a bottleneck point to prevent diverse, cell line-specific resistance programs.

Mediator kinase inhibition suppresses hyperactive interferon signaling in Down syndrome.

Hyperactive interferon (IFN) signaling is a hallmark of Down syndrome (DS), a condition caused by trisomy 21 (T21); strategies that normalize IFN signaling could benefit this population. Mediator-associated kinases CDK8 and CDK19 drive inflammatory responses through incompletely understood mechanisms. Using sibling-matched cell lines with/without T21, we investigated Mediator kinase function in the context of hyperactive IFN in DS over a 45min - 24h timeframe. Activation of IFN-response genes was suppressed in cells treated with the CDK8/CDK19 inhibitor cortistatin A (CA), and this occurred through rapid suppression of IFN-responsive transcription factor activity. Moreover, we discovered that CDK8/CDK19 affect splicing, a novel means by which Mediator kinases control gene expression. To further probe Mediator kinase function, we completed cytokine screens and untargeted metabolomics experiments. Cytokines are master regulators of inflammatory responses; by screening 105 different cytokine proteins, we show that Mediator kinases help drive IFN-dependent cytokine responses at least in part through transcriptional regulation of cytokine genes and receptors. Metabolomics revealed that Mediator kinase inhibition altered core metabolic pathways, including broad up-regulation of anti-inflammatory lipid mediators, whose levels were elevated during hyperactive IFN signaling. A subset of these lipid mediators (e.g. oleamide, desmosterol) serve as ligands for nuclear receptors PPAR and LXR, and activation of these receptors occurred specifically during hyperactive IFN signaling in CA-treated cells, revealing a mechanistic link between Mediator kinase activity and nuclear receptor function. Collectively, our results identify new mechanisms by which CDK8/CDK19 regulate gene expression, and establish that Mediator kinase inhibition antagonizes IFN signaling through transcriptional, metabolic, and cytokine responses, with implications for DS and other chronic inflammatory conditions.

Abstract LT04: Dissecting Mediator Kinase Activity in Normal Tissue Homeostasis and Cancer

Abstract Cyclin-dependent kinase 8 (CDK8) and its paralog CDK19, collectively known as Mediator kinase, are part of the multimeric Mediator complex that fine-tune gene expression programs on multiple levels, most notably by controlling transcription factor (TF) activity at promoter- and enhancer regions as well as organising the chromatin landscape. While Mediator kinases have been predominantly studied as a therapeutic target in cancer cells, their functions in a physiologically relevant context remain poorly understood. To study in vivo functions of Mediator, we developed a comprehensive suite of genetically engineered mouse models (GEMMs), including conditional floxed alleles (Cdk8fl/fl/Cdk19fl/fl) and kinase-dead knock-ins (Cdk8D173A/fl/Cdk19D173A/fl), enabling the analysis of scaffolding and kinase-dependent functions of this critical transcriptional module. Herein, we show that Mediator kinase has distinct kinase-dependent functions in modulating homeostasis and tissue lineage specification in diverse organs. Moreover, using a proteo-genomic approach we show that the Mediator Kinase module functionally interacts with the SWI/SNF complex to mediate transcriptional output. Collectively, our work expands the understanding of how Mediator Kinase regulates tissue homeostasis and provides a framework for considering how Mediator Kinase inhibition may be effectively employed in the clinical setting. Citation Format: Marius Dannappel, Danxi Zhu, Claire Sun, Ron Firestein. Dissecting Mediator Kinase Activity in Normal Tissue Homeostasis and Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT04.

Mediator kinase module proteins, genetic alterations and expression of super-enhancer regulated genes in colorectal cancer.

Genetic alterations are well characterized as contributors to the pathogenesis of cancers. Epigenetic abnormalities can lead to perturbations of the expression of genes in cancer cells without structural defects. Deregulation of proteins of the transcription machinery may result in perturbations of target genes. Mediator, a multiprotein component of the transcription machinery facilitates the function of RNA polymerase II, which transcribes most human genes. A part of the mediator with kinase activity, called the Mediator kinase module shows genetic alterations in a sub-set of colorectal cancers. Data from publicly available genomic series of colorectal cancer patients were examined to determine alterations of Mediator kinase module component genes, including MED12, MED12L, MED13, MED13L, CDK8, CDK19, and CCNC. The prevalence of alterations in genomically defined colorectal cancer sub-sets was also interrogated. The effect of Mediator kinase module member gene expression on colorectal cancer relapse-free survival was investigated. Mutations in genes of the Mediator kinase module were present in a small percentage of colorectal cancers, ranging between 2 to 10% for MED12 and MED13 and alternative units MED12L and MED13L and below 2% for kinases CDK8 and CDK19 and cyclin C. Amplifications of the CDK8 gene were observed in 3% to 5% of colorectal cancers. The highest prevalence of mutations was observed in MSI cancers and the equivalent CMS1 group, with other genomic groups showing much lower frequency. An association of higher expression of MED12 with inferior relapse-free survival was observed. In contrast, higher expression of cyclin C was associated with improved survival. Colorectal cancer cell lines with CDK8 amplifications displayed sensitivity to several small molecule inhibitors of the KRAS/PI3K pathway but not to BET inhibitors. The Mediator kinase module is deregulated in a sub-set of colorectal cancers with differences observed in genomically defined groups. These variations may result in differences in sensitivity to targeted therapies and may have to be taken into consideration as such therapies are developed.

Mediator kinase inhibition reverses castration resistance of advanced prostate cancer.

Mediator kinases CDK19 and CDK8, pleiotropic regulators of transcriptional reprogramming, are differentially regulated by androgen signaling, but both kinases are upregulated in castration-resistant prostate cancer (CRPC). Genetic or pharmacological inhibition of CDK8 and CDK19 reverses the castration-resistant phenotype and restores the sensitivity of CRPC xenografts to androgen deprivation in vivo. Prolonged CDK8/19 inhibitor treatment combined with castration not only suppressed the growth of CRPC xenografts but also induced tumor regression and cures. Transcriptomic analysis revealed that Mediator kinase inhibition amplified and modulated the effects of castration on gene expression, disrupting CRPC adaptation to androgen deprivation. Mediator kinase inactivation in tumor cells also affected stromal gene expression, indicating that Mediator kinase activity in CRPC molded the tumor microenvironment. The combination of castration and Mediator kinase inhibition downregulated the MYC pathway, and Mediator kinase inhibition suppressed a MYC-driven CRPC tumor model even without castration. CDK8/19 inhibitors showed efficacy in patient-derived xenograft models of CRPC, and a gene signature of Mediator kinase activity correlated with tumor progression and overall survival in clinical samples of metastatic CRPC. These results indicate that Mediator kinases mediated androgen-independent in vivo growth of CRPC, supporting the development of CDK8/19 inhibitors for the treatment of this presently incurable disease.

Abstract 3395: Polyisoprenylated cysteinyl amide inhibitors: Effects on RAS signaling pathway intermediates and viability of mutant KRAS African American lung cancer cell line

Abstract Mutated KRAS is the most common cancer driver gene in lung adenocarcinomas in all racial and ethnic groups, including Blacks/African Americans (B/AAs). Drugs developed to combat KRAS-driven cancers become less effective due to intrinsic changes in the cells resulting in resistance and relapse. Due to the enormous challenges encountered in developing direct KRAS inhibitors, agents targeting alternative downstream RAS mediator kinase enzymes of the MAPK and PI3K/AKT signaling pathways have been developed. However, other avenues that directly target KRAS and similar cancer drivers must be explored to obtain alternative effective therapies. PCAIs constitute a group of potential anticancer agents that we developed to specifically disrupt and suppress signaling by hyperactive G-proteins such as mutated KRAS. In previous studies, PCAIs stimulated MEK1/2, ERK1/2, p90RSK and AKT phosphorylation resulting in cell death. The present study aims to determine whether specific inhibitors of the kinases can reverse PCAIs-stimulated phosphorylation and cell death. PCAIs were thus tested against the KRAS mutant NCI- cell line H23 (from a B/AA patient) and compared to KRASG12C inhibitor Sotorasib and other kinase inhibitors drugs. The EC50 values for the PCAIs NSL-YHJ-2-27 and NSL-AB-45 were 4.1 ± 0.3 and 5.5 ± 0.1 μM, respectively, compared to Gefitinib (EGFR), Sotorasib (KRASG12C), Dabrafenib (BRAF), Selumetinib (MEK1/2), Ulixertinib (ERK1/2) and Ipatasertib (AKT) whose EC50 values were 83 ± 12.9 μM, 38 ± 5.5 μM, 33 ± 1.2 μM, 71 ± 5.3 μM, 11 ± 0.2 μM, 12 ± 1.0 μM, respectively. The results indicate that the PCAIs are more effective against NCI-H23 than the other kinase inhibitor drugs. Future work will determine the effects of co-treating cells with the PCAIs and the respective kinase inhibitors on cell viability and PCAIs-induced phosphorylation of the kinases. Supported by grants U54CA233396, U54CA233444, and U54CA233465 from the National Institutes of Health (NIH)/National Cancer Institute (NCI) and the Norris Comprehensive Cancer Center core grant, award number P30CA014089 from the NIH/NCI Citation Format: Justin K. Mensah-Mamfo, Kweku Ofosu-Asante, Jassy Mary Lazarte, Amarender Goud Burra, Sofia A. Lugo, Yong Huang, Ite A. Offringa, Nazarius S. Lamango. Polyisoprenylated cysteinyl amide inhibitors: Effects on RAS signaling pathway intermediates and viability of mutant KRAS African American lung cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3395.

Abstract B058: Targeting mediator kinase CDK8/19 potentiates chemotherapeutic responses, reverses tumor growth, and prolongs survival from ovarian clear cell carcinoma

Abstract Ovarian clear cell carcinomas (OCCC) are a rare histological subtype of epithelial ovarian cancer with known treatment resistance, a feature associated with transcriptional reprogramming. CDK8 and CDK19 Mediator Kinases, which are isoforms of the Mediator Kinase – enzymatic components of a regulatory module that binds to the transcriptional Mediator complex –and not a part of the overall transcription machinery, have been previously reported to modify oncogenic transcription factors. Inhibition of the kinase activity of CDK8/19 was shown to prevent and reverse chemoresistance in other cancers with two CDK8/19 inhibitors currently in clinical trials for non–ovarian cancers. Based on the unmet need for OCCCs, we assessed CDK8/19 expression in a panel of OCCC primary and metastatic tissues and utilized cell line models for investigation into the therapeutic potential of CDK8/19 inhibitors in preventing tumorigenesis and synergizing with existing chemotherapeutics. The synergistic effects of SNX631-6, a preclinical CDK8/19 inhibitor drug candidate, were tested in vitro using three clear cell (ES2, TOV-21-G, RMG1) and one non-clear cell (SKOV3) ovarian carcinoma cell lines. A sub-inhibitory concentration 50 (IC50) concentration of SNX631-6 (500 nM) strongly potentiated effects of cisplatin, carboplatin, and paclitaxel. Additionally, cells demonstrating platinum resistance reverted to platinum sensitivity when exposed to the same concentration of SNX631-6. In vivo antitumor effects and tolerability of oral SNX631-6 were assessed in Foxn1nu homozygous female mice injected subcutaneously with ES2+luc cells. Mice were treated with a vehicle (n=10), intraperitoneal (IP) cisplatin (2 mg/kg) twice weekly (n=9), SNX631-6 medicated diet (330 ppm) orally ad libitum (n=10), or IP cisplatin combined with SNX631 diet (n=10) until tumors reached a predetermined critical volume of 2500mm3. In ES2+luc injected mice, significant tumor growth inhibition relative to vehicle occurred in mice treated with a combination of IP cisplatin and SNX631-6 (p<0.001) or SNX631-6 alone (p<0.01). Additionally, the use of IP cisplatin and SNX631-6 significantly delayed time-to-critical-volume compared to vehicle (p=0.0001). SNX631-6 alone and in combination with IP cisplatin was well tolerated in all mice based on body weight measurements. In summary, SNX631-6 potentiated platinum responses in vitro and demonstrated strong in vivo antitumor effects, including tumor regression, when used as monotherapy and in combination with platinum-based therapy. These data suggest that therapeutically achievable doses of SNX631-6 may provide clinical benefit, prevent tumorigenesis, and reverse platinum resistance in patients with OCCC. Citation Format: Wade Barton, Asha Kumari, Gary School, Zachary Mack, Liz Macias Quintero, Karthik Rangavajhula, Rebecca Arend, Mengqian Chen, Eugenia V. Broude, Karthikeyan Mythreye. Targeting mediator kinase CDK8/19 potentiates chemotherapeutic responses, reverses tumor growth, and prolongs survival from ovarian clear cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B058.

Abstract A019: Development of adaptive anoikis resistance promotes ovarian cancer metastasis that can be prevented by CDK8/19 Mediator kinase inhibition

Abstract Anoikis resistance or evasion of cell death triggered by cell detachment is a hallmark of cancer that is concurrent with cell survival and metastasis. Phenotypes of intrinsically anoikis resistant cancer cells have been extensively investigated but the mechanism of how exposure to suspension stress may lead to the acquisition of an anoikis resistance phenotype has not been previously described. Here we show that cycles of suspension stress followed by attached growth, mimicking an in vivo scenario, lead to adaptation to anoikis and acquisition of resistance to cell death, concomitant with increased chemoresistance and metastasis in vivo. Based on mutational and clonal population behavior analysis and modeling using a modified Luria-Delbrück’s fluctuation analysis we find that such acquired anoikis resistance is not genetic. Transcriptional reprogramming is, however,essential for the acquisition of adaptive anoikis resistance in vitro and in vivo, since these processes are exquisitely sensitive to selective inhibition of the CDK8/19 Mediator kinase, a broad-spectrum regulator of transcriptional reprogramming. Longitudinal analysis of the acquisition of anoikis resistance indicates strong repression programs along with a hybrid epithelial/mesenchymal phenotype and an enhanced need for oxidative phosphorylation, resulting in the emergence of anoikis-adapted cells that also efficiently evade T cell-mediated immune surveillance. Our data demonstrate that recovery from the brink of anoikis is a direct contributor to metastasis in ovarian cancer and can be therapeutically prevented using inhibitors of CDK8/19 Mediator kinase Citation Format: Mehri Monavarian, Resha Rajkarnikar, Emily Faith Page, Asha Kumari, Liz Macias Quintero, Lara Ianov, sarthak Sahoo, Mohit Kumar Jolly, Elizabeth Worthey, Nadine Hempel, Abhyudai Singh, Eugenia Broude, Karthikeyan Mythreye. Development of adaptive anoikis resistance promotes ovarian cancer metastasis that can be prevented by CDK8/19 Mediator kinase inhibition [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A019.

Mediator kinase subunit CDK8 phosphorylates transcription factor TCP15 during tomato pollen development.

Pollen development in flowering plants has strong implications for reproductive success. Pollen DNA can be targeted to improve plant traits for yield and stress tolerance. In this study, we demonstrated that the Mediator subunit CYCLIN-DEPENDENT KINASE 8 (CDK8) is a key modulator of pollen development in tomato (Solanum lycopersicum). SlCDK8 knockout led to significant decreases in pollen viability, fruit yield, and fruit seed number. We also found that SlCDK8 directly interacts with transcription factor TEOSINTE BRANCHED1-CYCLOIDEA-PCF15 (SlTCP15) using yeast two-hybrid screens. We subsequently showed that SlCDK8 phosphorylates Ser 187 of SlTCP15 to promote SlTCP15 stability. Phosphorylated TCP15 directly bound to the TGGGCY sequence in the promoters of DYSFUNCTIONAL TAPETUM 1 (SlDYT1) and MYB DOMAIN PROTEIN 103 (SlMYB103), which are responsible for pollen development. Consistently, disruption of SlTCP15 resembled slcdk8 tomato mutants. In sum, our work identified a new substrate of Mediator CDK8 and revealed an important regulatory role of SlCDK8 in pollen development via cooperation with SlTCP15.

The Mediator kinase module enhances polymerase activity to regulate transcriptional memory after heat stress in Arabidopsis

Plants are often exposed to recurring adverse environmental conditions in the wild. Acclimation to high temperatures entails transcriptional responses, which prime plants to better withstand subsequent stress events. Heat stress (HS)-induced transcriptional memory results in more efficient re-induction of transcription upon recurrence of heat stress. Here, we identified CDK8 and MED12, two subunits of the kinase module of the transcription co-regulator complex, Mediator, as promoters of heat stress memory and associated histone modifications in Arabidopsis. CDK8 is recruited to heat-stress memory genes by HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2). Like HSFA2, CDK8 is largely dispensable for the initial gene induction upon HS, and its function in transcriptional memory is thus independent of primary gene activation. In addition to the promoter and transcriptional start region of target genes, CDK8 also binds their 3ʹ-region, where it may promote elongation, termination, or rapid re-initiation of RNA polymerase II (Pol II) complexes during transcriptional memory bursts. Our work presents a complex role for the Mediator kinase module during transcriptional memory in multicellular eukaryotes, through interactions with transcription factors, chromatin modifications, and promotion of Pol II efficiency.

Abstract A043: CDK8/19-regulated transcriptional reprogramming: a druggable driver of castration-resistant prostate cancer

Abstract Androgen deprivation therapy (ADT) is the mainstay of prostate cancer (PCa) treatment. However, most patients with aggressive PCa become resistant to ADT and develop castration-resistant prostate cancer (CRPC). Newer androgen receptor (AR)-targeting CRPC treatments extend survival by only a few months, and metastatic CRPC (mCRPC) remains an incurable disease. Multiple mechanisms of ADT resistance have been identified, among which non-genetic (transcriptional) mechanisms are prominent, due to the high transcriptomic plasticity of PCa and the role of tumor microenvironment (TME) as a determinant of ADT resistance. PCa progression involves changes in the expression of CDK8 and CDK19, enzymatic components of the cyclin-dependent kinase module (CKM), associated with the transcriptional Mediator complex and implicated in transcriptional reprogramming and immune surveillance. We have investigated the roles of CDK8/19 Mediator kinases in CRPC, through bioinformatic analysis of primary PCa and mCRPC and by analyzing the effects of genetic and pharmacological inhibition of CDK8 and CDK19 on tumor growth and gene expression. CDK8 and CDK19 were found to be differentially regulated by androgen, which downregulates CDK8 and upregulates CDK19. Accordingly, CDK8 is decreased and CDK19 increased in primary PCa, where CDK19 is expressed higher than in any other tissues or tumors. However, both CDK19 and CDK8, as well as the other components of the CKM, are upregulated in mCRPC, concordantly with the changes in AR-mediated transcriptional signaling. Selective Mediator kinase inhibitors did not suppress PCa models driven by canonical AR signaling but inhibited in vivo growth of cell line-based and patient-derived xenograft (PDX) models of CRPC. Genetic inactivation of CDK8 and CDK19 suppressed 22Rv1 CRPC tumors in surgically or chemically castrated (but not intact) male NSG mice, whereas the expression of CDK19 or CDK8 reversed this phenotype. Transcriptomic analysis revealed that Mediator kinase inhibition exerted the strongest effects on gene expression in tumors growing in castrated mice, increasing the transcriptomic effects of castration on most of the genes regulated by CDK8/19, while decreasing its effects on some of the genes implicated in PCa progression. A gene signature reflecting Mediator kinase activity in CRPC xenografts correlated with shorter overall survival among mCRPC patients. Systemic treatment with a CDK8/19 inhibitor also affected stromal gene expression; many of these effects were reproduced by Mediator kinase mutagenesis in tumor cells alone, suggesting that Mediator kinases of tumor cells are involved in shaping the TME. Prolonged (up to 300 days) CDK8/19 inhibition induced not only growth inhibition but also shrinkage of CRPC tumors in nude mice (which contain NK cells that are stimulated by CDK8/19 inhibition) and even produced cures in 18-27% of animals. These results warrant the exploration of CDK8/19 Mediator kinase inhibitors for the treatment of the presently incurable CRPC. Citation Format: Jing Li, Thomas Hilimire, Yueying Liu, Lili Wang, Jiaxin Liang, Balazs Gyorffy, Vitali Sikitzhytski, Hao Ji, Li Zhang, Chen Cheng, Xiaokai Ding, Kendall Kerr, Charles Dowling, Gary Schools, Chang-Uk Lim, Eugenia Broude, George Wilding, Michael Lilly, Igor Roninson, Mengqian Chen. CDK8/19-regulated transcriptional reprogramming: a druggable driver of castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A043.

Mediator Kinase/CDK8 Inhibition As a Strategy to Improve FLT3 Inhibitor Activity in Acute Myeloid Leukemia

Acquired resistance to targeted FLT3 tyrosine kinase inhibitors (TKIs) remains a major barrier to durable clinical responses for patients with FLT3-mutant AML. Multiple resistance mechanisms to next generation FLT3 TKIs converge upon RAS/MAPK pathway activation, including protective cytokine stimuli of the bone marrow microenvironment or through secondary oncogenic RAS mutations. RAS mutations also decrease efficacy of the BCL2 inhibitor venetoclax now widely used in AML, suggesting an anti-apoptotic influence of MAPK signaling. These findings highlight a significant unmet need for novel therapeutic combinations to address MAPK-mediated resistance in AML. We report results from a genome-wide CRISPR interference (CRISPRi) apoptosis screen using FLT3-ITD mutant MOLM-14 cells treated with the FLT3 TKI gilteritinib while cultured in HS5 conditioned media (CM) to mimic cell-extrinsic MAPK-driven TKI resistance. We identified RNA pol II and Mediator kinase/CDK8 ascritical nodes for AML cell survival in the setting of FLT3 inhibition. We further demonstrated that CDK8 inhibition with the small-molecule SEL120 re-sensitized multiple FLT3-mutant AML cell lines and primary samples to gilteritinib. We also performed a second CRISPRi apoptosis screen to identify hits that sensitize to gilteritinib in a model of cell-intrinsic MAPK-mediated resistance using MOLM-14 cells that harbor secondary NRAS-G12C mutation.We used Enrichr to perform an integrated analysis and validated the Core Mediator Complex and CDK8 knockdown genes as cellular components significantly enriched among sensitizing hits in both screens. The Mediator subunit MED12 was identified as a top gene level hit that strongly sensitized cells to gilteritinib in both screens, further emphasizing that Mediator kinase and its transcriptional regulation may be a central pathway engaged in MAPK-dependent survival in FLT3-mutant AML. To better understand potential mechanisms of increased combinatorial CDK8i/FLT3i activity, we performed RNA-seq in MOLM-14 cells treated with gilteritinib, SEL120, or the combination in HS5 CM. Using GSEA we found that gilteritinib stimulated adaptive interferon/inflammatory gene signatures at 16h drug treatment, but this response was restrained by addition of SEL120. Additionally, we identified a striking upregulation of the lineage-controlling transcription factor IRF8 and its gene expression signature with concomitant silencing of SPI1 transcriptional output in cells treated with gilteritinib/SEL120. CDK8i treatment alone in AML has previously been shown to increase expression of super-enhancer (SE) associated genes, including IRF8. Interestingly, SPI1was also a significant gene-level hit in both CRISPRi screens. Using CRISPRi we repressed IRF8 expression in MOLM-14, MOLM-14 NRAS-G12C, and MV411 (another FLT3-mutant line) cells and found this significantly blunted the combinatorial pro-apoptotic effect of SEL120, implying that induction of IRF8 expression plays a significant role in driving response to combined gilteritinib/SEL120. We finally assessed in vivo activity of gilteritinib combined with SEL120. We first performed pilot toxicity studies in NSG mice and determined that gilteritinib 30 mg/kg, SEL120 30 mg/kg, or the combination dosed 5 days/week by oral gavage (OG) were regimens well tolerated by mice. We then engrafted luciferase-tagged MOLM-14 and MOLM-14 NRAS-G12C cells into NSG mice and treated for 4-weeks with gilteritinib, SEL120, or the combination. We found gilteritinib/SEL120 extended survival in both MOLM-14 and MOLM-14 NRAS-G12C models. Similar efficacy studies are ongoing in FLT3-mutant patient-derived xenograft (PDX) models with NSGS mice and will be reported. Our results indicate that FLT3-mutant AML cells employ an adaptive inflammatory response to evade FLT3i-induced apoptosis, and CDK8i represses this adaptation while concomitantly increasing the ratio of IRF8 to SPI1-driven gene expression programs. We speculate this latter effect may in part alter an AML cell's differentiation state to sensitize cells to apoptosis, though further study to explore this hypothesis is needed. Our in vitro and in vivo efficacy data further validated combined FLT3i/CDK8i as a promising investigational strategy to pre-empt or overcome MAPK-mediated FLT3 TKI resistance.

Novel Clinically Useful Inhibitor of Mediator Complex, RVU120, Relives Differentiation Block in MDS/AML

Background: Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemias (AML) are characterized by a block in hematopoietic differentiation that can often be traced to aberrant stage-specific transcription. The kinase module of the mediator complex, consisting of CDK8, its paralog CDK19, Cyclin C, MED12, and MED13, have been identified as a molecular switch that regulates gene expression. A variety of cancers have exploited this mechanism to maintain stemness and an undifferentiated state. RVU120 is a first-in-class, specific, and selective inhibitor of CDK8/19, currently in Phase Ib clinical trial in patients with AML or High-Risk MDS (HR-MDS) (NCT04021368). RVU120 has been shown to stimulate erythroid differentiation in transformed CD34+ cord blood (CB) cells and in CD34+ cells from Diamond-Blackfan Anemia (DBA). To evaluate the further therapeutic potential of CDK8/19 inhibition, we confirmed aberrant expression of mediator complex components and erythroid-stimulating activity in a collection of CD34+ primary samples of AML and MDS. Results: We investigated the expression of mediator complex components in a large database of MDS CD34+ marrow samples and age-matched controls. Our findings revealed that MED12, a critical component of the mediator complex, was significantly overexpressed in MDS samples from refractory anemia with excess blasts (RAEB), a higher risk subset of MDS(p=0.018) (Fig A). Furthermore, we observed that this overexpression of MED12 was associated with a higher rate of transformation to AML. To delve deeper, we examined the expression of MED12 in sorted leukemic stem cells from primary AML samples and found significantly elevated levels in Long Term Hematopoietic Stem Cells from AML samples with both normal karyotype and complex karyotypes compared to healthy controls. Next, we conducted an evaluation of the efficacy of the mediator kinase module inhibition in MDS-derived cell line (MDS-L). Inhibition of CDK8/19 with RVU120 led to increased erythroid differentiation in this model which confirmed our observations in transformed CD34+ CB cells in CB cells, these phenotypic changes were correlated with the inhibition of CDK8-dependent phosphorylation marker S726 on STAT5 and a profound increase in the expression of genes involved in erythroid commitment and hemoglobin metabolism. In order to confirm therapeutic potential of CDK8/CDK19 inhibition in MDS and AML, we performed colony-forming assay on primary samples of MDS and AML (n= 15). The treatment with RVU120 resulted in increased erythroid differentiation in a majority of samples, evident from an augmented number of colonies. Detailed analysis indicated changes in the expression of erythroid differentiation markers, including increased CD71 and Glycophorin A expression on colonies, as assessed by FACS analysis (Figure B, shows representative sample). Conclusion Our data highlight that inhibition of the overexpressed mediator complex proteins can alleviate the differentiation blocks seen in MDS/AML. These results provide a compelling rationale for further developing the CDK8/19 inhibitor RVU120 for transfusion- dependent MDS/AML patients.

CRISPR Activation Screen to Optimize Chimeric Antigen Receptor (CAR) T Cell Immunophenotype

Background Adoptive cellular therapies (ACT) offer potential cures for a wide range of human diseases. Immune cells genetically engineered with chimeric antigen receptors (CAR) have a clear role in B cell malignancies, and broader applications to other malignancies are under investigation. Significant obstacles to the long-term durability and potency of these therapies remain. As CAR T-cells remain persistently activated and exposed to the tumor microenvironment, they may become functionally impaired. This exhaustion can result in reduced production of cytokines, cytotoxic molecules, and other factors necessary for effective tumor cell killing. While CAR T-cell exhaustion remains a challenge, ongoing research and clinical trials aim to further understand the underlying mechanisms and develop strategies to overcome this limitation. Clinical studies have demonstrated that sustained in vivo persistence and function of CAR T-cells is correlated with infusion product phenotype. Persistence is associated with a less differentiated, memory-like phenotype. Naive (T N), Central Memory (T CM) and Stem Cell Memory (T SCM) are related to good proliferative potential and durability. CRISPR technologies offer powerful, functional genomic screens that permit the inference and modification of gene regulatory networks to optimize CAR T-cell persistence and function. Genome-wide CRISPR knock out screens have identified the CD2-CD58 axis as critical to T-cell potency and the knock-out of the mediator kinase module as beneficial to potency. Here we report on in vitro CRISPR activation screening of human CAR T-cells for gene expression that enhances T cell phenotypes associated with durable function. Method We designed 9,983 single guide RNA (sgRNA) to target the transcription start sites (TSS) of all known human transcription factors as well as a select set of genes with critical functions in T-cells. The vast majority of genes was targeted with 5 different sgRNA. To this we added a pool of 1,000 non-targeting sgRNA. We lentivirally transduced the sgRNA in tandem to a second-generation anti-CD20 CAR into primary T cells from healthy human donors. In a second lentiviral transduction we integrated catalytically dead Cas9 (dCas9) fused to the strong transcription activator, VP64 and in tandem to a puromycin resistance gene. After puromycin selection, we cultured the CRISPR activated CAR T-cells either alone or together with Raji or K562 cells for nine days before immunophenotypically sorting the CAR T-cells into CD8 + Naive, CD8 + Transitional Memory, CD8 + Central Memory, CD8 + Stem Cell Memory, CD8 + Transitional Effector, CD8 + Effector Memory, CD4 + Naive and CD4 + Memory T-cell populations. We extracted DNA from the sorted cells and prepared sequencing libraries targeting the sgRNA. We carried out two-way ANOVA analysis on the experimental conditions (trivial, Raji or K562 co-culture versus addition of CD2/CD28 activation beads) and T-cell subset to determine which genes targeted by CRISPRa resulted in favorable phenotypes. Result Across the different culture conditions, sgRNA activating IL23A and KLF4 were enriched in Transitional Memory versus Stem Cell Memory T-cells. Across the different CAR T-cell immunophenotypes, sgRNA targeting the TGFß (including KLF10), TNFα (including NFATC4) and FOXO signaling pathways were enriched in CAR T-cells cultured alone compared to CAR T-cells activated with CD2/CD28. Conclusion In this study we have investigated genes whose activation results in CAR T-cell immunophenotypes that are associated with more potent and durable CAR T-cell therapies. Our experiment suggests that the activation of genes, such as IL23 and KLF4, enriches for a Transitional Memory over Stem Cell Memory phenotype in a manner that is largely independent of environment (culture conditions). The activation of other genes, such as KLF10 and NFATC4, results in enrichment of CAR T-cells across all T-cell subsets, but in a manner that is dependent on environment. Gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), whose state varies as cells differentiate or respond to environmental signals. Abrogating or adding CRE may result in more durable and potent adoptive cellular therapies independent of environment.

RISING STARS: Role of MED12 mutation in the pathogenesis of uterine fibroids.

Uterine fibroids (UFs) are benign tumors arising from the uterus, characterized by accumulation of abundant extracellular matrix (ECM) and sex steroid-dependent growth. Women with symptomatic UFs have reduced quality of life and decreased labor productivity. Among the driver gene mutations identified in UFs, mutations in MED12, a component of the cyclin-dependent kinase (CDK) Mediator module, are the most common and observed in 50-80% of UFs. They are gain-of-function mutations and are more frequently observed in Black women and commonly observed even in small UFs. MED12 mutation-positive UFs (MED12-UFs) often develop multiple rather than solitary and have distinct gene expression profiles, DNA methylomes, transcriptomes, and proteomes. Gene expressions related to ECM organization and collagen-rich ECM components are upregulated, and impaired Mediator kinase activity and dysregulation of Wnt/β-catenin signaling are identified in MED12-UFs. Clinically, the UF shrinking effect of gonadotropin-releasing hormone agonists and ulipristal acetate is dependent on the MED12 mutation status. Understanding of characteristics of MED12-UFs and functions of MED12 mutations for UF tumorigenesis may elucidate the pathophysiology of UFs, leading to the development of new therapeutic options in women with symptomatic UFs.

Abstract 2012: Mediator kinase inhibition suppresses chronic inflammation in Down syndrome

Abstract 2012: Mediator kinase inhibition suppresses chronic inflammation in Down syndrome

Abstract 2644: Mediator kinase disruption links altered myometrial stem cell enhancer dynamics with uterine fibroid formation

Abstract 2644: Mediator kinase disruption links altered myometrial stem cell enhancer dynamics with uterine fibroid formation

Enhanced T cell Effector Activity by Targeting the Mediator Kinase Module

T cells play important roles in combating various diseases, including cancers. To find out what genes hinder the function of T cells, Freitas and colleagues performed genome-wide CRISPR knock-out experiments in human chimeric antigen receptor T cells. They found that two genes, MED12 and CCNC, which are part of the Mediator kinase module, had the most significant effect. They discovered that antitumor activity was increased when MED12 was selectively deleted, and the effector phenotype was maintained in chimeric antigen receptor and T cell receptor-engineered T cells. They found increased occupancy of core Mediator chromatin at transcriptionally active enhancers in MED12-deficient cells. This effect was most evident for enhanced IL2RA expression and STAT and AP-1 transcription factors. The authors concluded that Mediator plays a vital role in T cell effector programming and that antitumor T cell response may be enhanced by targeting the Mediator kinase module. Science (2022). DOI: 10.1126/science.abn5647.