ERK5 Interacts with Mitochondrial Glutaminase and Regulates Its Expression.

Many of the biological processes of the cell, from its structure to signal transduction, involve protein-protein interactions. On this basis, our aim was to identify cellular proteins that interact with ERK5, a serine/threonine protein kinase with a key role in tumor genesis and progression and a promising therapeutic target in many tumor types. Using affinity chromatography, immunoprecipitation, and mass spectrometry techniques, we unveiled an interaction between ERK5 and the mitochondrial glutaminase GLS in pancreatic tumor cells. Subsequent co-immunoprecipitation and immunofluorescence studies supported this interaction in breast and lung tumor cells as well. Genetic approaches using RNA interference techniques and CRISPR/Cas9 technology demonstrated that the loss of ERK5 function led to increased protein levels of GLS isoforms (KGA/GAC) and a concomitant increase in their activity in tumor cells. It is well known that the tumor cell reprograms its intermediary metabolism to meet its increased metabolic needs. In this sense, mitochondrial GLS is involved in the first step of glutamine catabolism, one of the main energy sources in the context of cancer. Our data suggest that ERK5 contributes to the regulation of tumor cell energy metabolism via glutaminolysis.

De Novo Missense Substitutions in the Gene Encoding CDK8, a Regulator of the Mediator Complex, Cause a Syndromic Developmental Disorder.

The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in MED12, MED13, and MED13L were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report CDK8 mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense CDK8 substitutions, including 10 shown to have arisen de novo, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a CDK8 and CDK19 CRISPR double-knockout cell line transfected with wild-type (WT) or mutant CDK8 constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in CDK8 cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms.

Assessing the mechanism and therapeutic potential of modulators of the human Mediator complex-associated protein kinases.

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.

Structure-Based Optimization of Potent, Selective, and Orally Bioavailable CDK8 Inhibitors Discovered by High-Throughput Screening.

The mediator complex-associated cyclin dependent kinase CDK8 regulates ß-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies.

Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19.

The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.

A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease.

There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen.

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

Therapeutic potential of ERK5 targeting in triple negative breast cancer.

Triple negative breast cancers (TNBCs) account for 15% of all breast cancers, and represent one of the most aggressive forms of the disease, exhibiting short relapse-free survival. In contrast to other breast cancer subtypes, the absence of knowledge about the etiopathogenic alterations that cause TNBCs force the use of chemotherapeutics to treat these tumors. Because of this, efforts have been devoted with the aim of incorporating novel therapies into the clinical setting. Kinases play important roles in the pathophysiology of several tumors, including TNBC. Since expression of the MAP kinase ERK5 has been linked to patient outcome in breast cancer, we analyzed the potential value of its targeting in TNBC. ERK5 was frequently overexpressed and active in samples from patients with TNBC, as well as in explants from mice carrying genetically-defined TNBC tumors. Moreover, expression of ERK5 was linked to a worse prognosis in TNBC patients. Knockdown experiments demonstrated that ERK5 supported proliferation of TNBC cells. Pharmacological inhibition of ERK5 with TG02, a clinical stage inhibitor which targets ERK5 and other kinases, inhibited cell proliferation by blocking passage of cells through G1 and G2, and also triggered apoptosis in certain TNBC cell lines. TG02 had significant antitumor activity in a TNBC xenograft model in vivo, and also augmented the activity of chemotherapeutic agents commonly used to treat TNBC. Together, these data indicate that ERK5 targeting may represent a valid strategy against TNBC, and support the development of trials aimed at evaluating the clinical effectiveness of drugs that block this kinase.

Potent antimyeloma activity of a novel ERK5/CDK inhibitor.

PURPOSE: To analyze the antimyeloma potential of TG02, an ERK5/CDK inhibitory drug. EXPERIMENTAL DESIGN: Utilizing different multiple myeloma cell lines we determined the effect of TG02 over viability by MTT assays. The apoptotic effect over multiple myeloma patient samples was studied ex vivo by cytometry. The mechanism of action of TG02 was analyzed in the cell line MM1S, studying its effect on the cell cycle, the induction of apoptosis, and the loss of mitochondrial membrane potential by cytometry and Western blot. Two models of multiple myeloma xenograft were utilized to study the in vivo action of TG02. RESULTS: TG02 potently inhibited proliferation and survival of multiple myeloma cell lines, even under protective bone marrow niche conditions, and selectively induced apoptosis of primary patient-derived malignant plasma cells. TG02 displayed significant single-agent activity in two multiple myeloma xenograft models, and enhanced the in vivo activity of bortezomib and lenalidomide. Signaling analyses revealed that the drug simultaneously blocked the activity of CDKs 1, 2, and 9 as well as the MAP kinase ERK5 in MM1S cells, leading to cell-cycle arrest and rapid commitment to apoptosis. TG02 induced robust activation of both the intrinsic and extrinsic pathways of apoptosis, and depletion of XIAP and the key multiple myeloma survival protein Mcl-1. CONCLUSIONS: TG02 is a promising new antimyeloma agent that is currently in phase I clinical trials in leukemia and multiple myeloma patients.

Expression of Erk5 in early stage breast cancer and association with disease free survival identifies this kinase as a potential therapeutic target.

BACKGROUND: Breast cancer is the most common neoplasia in women. Even though advances in its treatment have improved disease outcome, some patients relapse. Therefore, attempts to better define the molecular determinants that drive breast cancer cell proliferation may help in defining potential therapeutic targets. Mitogen-activated protein kinases (MAPK) play important roles in tumorigenesis. One of them, Erk5, has been linked to the proliferation of breast cancer cells in vitro. Here we have investigated the expression and prognostic value of Erk5 in human breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: Animal and cellular models were used to study Erk5 expression and function in breast cancer. In 84 human breast tumours the expression of Erk5 was analyzed by immunohistochemistry. Active Erk5 (pErk5) was studied by Western blotting. Correlation of Erk5 with clinicopathological parameters and with disease-free survival in early stage breast cancer patients was analyzed. Expression of Erk5 was detected in most patients, and overexpression was found in 20%. Active Erk5 was present in a substantial number of samples, as well as in tumours from an animal breast cancer model. Overexpression of Erk5 was associated with a decrease in disease-free survival time, which was independent of other clinicopathological parameters of prognosis. Transient transfection of a short hairpin RNA (shRNA) targeting Erk5, and a stable cell line expressing a dominant negative form of Erk5 (Erk5(AEF)), were used to investigate the influence of Erk5 on drugs used in the clinic to treat breast tumours. We found that inhibition of Erk5 decreased cancer cell proliferation and also sensitized these cells to the action of anti-HER2 therapies. CONCLUSIONS/SIGNIFICANCE: Overexpression of Erk5 is an independent predictor of disease-free survival in breast cancer, and may represent a future therapeutic target.