Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics

Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-Seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0 – 24h), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair.Chemistry and Chemical Biolog

Mediator Kinase Inhibition Further Activates Super-Enhancer Associated Genes in AML

Super-enhancers (SEs), which are composed of large clusters of enhancers densely loaded with the Mediator complex, transcription factors (TFs), and chromatin regulators, drive high expression of genes implicated in cell identity and disease, such as lineage-controlling TFs and oncogenes 1, 2. BRD4 and CDK7 are positive regulators of SE-mediated transcription3,4,5. In contrast, negative regulators of SE-associated genes have not been well described. Here we report that Mediator-associated kinases cyclin-dependent kinase 8 (CDK8) and CDK19 restrain increased activation of key SE-associated genes in acute myeloid leukaemia (AML) cells. We determined that the natural product cortistatin A (CA) selectively inhibited Mediator kinases, had antileukaemic activity in vitro and in vivo, and disproportionately induced upregulation of SE-associated genes in CA-sensitive AML cell lines but not in CA-insensitive cell lines. In AML cells, CA upregulated SE-associated genes with tumour suppressor and lineage-controlling functions, including the TFs CEBPA, IRF8, IRF1 and ETV6 6, 7, 8. The BRD4 inhibitor I-BET151 downregulated these SE-associated genes, yet also has antileukaemic activity. Individually increasing or decreasing expression of these TFs suppressed AML cell growth, providing evidence that leukaemia cells are sensitive to dosage of SE-associated genes. Our results demonstrate that Mediator kinases can negatively regulate SE-associated gene expression in specific cell types and can be pharmacologically targeted as a therapeutic approach to AML

Diels–Alder Cycloaddition for Fluorophore Targeting to Specific Proteins inside Living Cells

The inverse-electron-demand Diels–Alder cycloaddition between <i>trans</i>-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. <i>Escherichia coli</i> lipoic acid ligase site-specifically ligates a <i>trans</i>-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine–fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels–Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase

NVL-520 Is a Selective, TRK-Sparing, and Brain-Penetrant Inhibitor of ROS1 Fusions and Secondary Resistance Mutations

ROS1 tyrosine kinase inhibitors (TKI) have been approved (crizotinib and entrectinib) or explored (lorlatinib, taletrectinib, and repotrectinib) for the treatment of ROS1 fusion–positive cancers, although none of them simultaneously address the need for broad resistance coverage, avoidance of clinically dose-limiting TRK inhibition, and brain penetration. NVL-520 is a rationally designed macrocycle with &gt;50-fold ROS1 selectivity over 98% of the kinome tested. It is active in vitro against diverse ROS1 fusions and resistance mutations and exhibits 10- to 1,000-fold improved potency for the ROS1 G2032R solvent-front mutation over crizotinib, entrectinib, lorlatinib, taletrectinib, and repotrectinib. In vivo, it induces tumor regression in G2032R-inclusive intracranial and patient-derived xenograft models. Importantly, NVL-520 has an ∼100-fold increased potency for ROS1 and ROS1 G2032R over TRK. As a clinical proof of concept, NVL-520 elicited objective tumor responses in three patients with TKI-refractory ROS1 fusion–positive lung cancers, including two with ROS1 G2032R and one with intracranial metastases, with no observed neurologic toxicities.Significance:The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion–positive patients.</p

NVL-520 Is a Selective, TRK-Sparing, and Brain-Penetrant Inhibitor of ROS1 Fusions and Secondary Resistance Mutations

ROS1 tyrosine kinase inhibitors (TKI) have been approved (crizotinib and entrectinib) or explored (lorlatinib, taletrectinib, and repotrectinib) for the treatment of ROS1 fusion–positive cancers, although none of them simultaneously address the need for broad resistance coverage, avoidance of clinically dose-limiting TRK inhibition, and brain penetration. NVL-520 is a rationally designed macrocycle with &gt;50-fold ROS1 selectivity over 98% of the kinome tested. It is active in vitro against diverse ROS1 fusions and resistance mutations and exhibits 10- to 1,000-fold improved potency for the ROS1 G2032R solvent-front mutation over crizotinib, entrectinib, lorlatinib, taletrectinib, and repotrectinib. In vivo, it induces tumor regression in G2032R-inclusive intracranial and patient-derived xenograft models. Importantly, NVL-520 has an ∼100-fold increased potency for ROS1 and ROS1 G2032R over TRK. As a clinical proof of concept, NVL-520 elicited objective tumor responses in three patients with TKI-refractory ROS1 fusion–positive lung cancers, including two with ROS1 G2032R and one with intracranial metastases, with no observed neurologic toxicities.Significance:The combined preclinical features of NVL-520 that include potent targeting of ROS1 and diverse ROS1 resistance mutations, high selectivity for ROS1 G2032R over TRK, and brain penetration mark the development of a distinct ROS1 TKI with the potential to surpass the limitations of earlier-generation TKIs for ROS1 fusion–positive patients.</p

NVL-520 is a selective, TRK-sparing, and brain-penetrant inhibitor of ROS1 fusions and secondary resistance mutations.

ROS1 tyrosine kinase inhibitors (TKIs) have been approved (crizotinib and entrectinib) or explored (lorlatinib, taletrectinib, and repotrectinib) for the treatment of ROS1 fusion-positive cancers, although none simultaneously address the need for broad resistance coverage, avoidance of clinically dose-limiting TRK inhibition, and brain penetration. NVL-520 is a rationally designed macrocycle with \u3e50-fold ROS1 selectivity over 98% of the kinome tested. It is active in vitro against diverse ROS1 fusions and resistance mutations and exhibits 10-to-1,000-fold improved potency for the ROS1 G2032R solvent-front mutation over crizotinib, entrectinib, lorlatinib, taletrectinib, and repotrectinib. In vivo, it induces tumor regression in G2032R-inclusive intracranial and patient-derived xenograft models. Importantly, NVL-520 has a ~100-fold increased potency for ROS1 and ROS1 G2032R over TRK. As clinical proof-of-concept, NVL-520 elicited objective tumor responses in three patients with TKI-refractory ROS1 fusion-positive lung cancers, including two with ROS1 G2032R and one with intracranial metastases, with no observed neurological toxicities