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.

No abstract

The cortistatins are a recently identified class of marine natural products characterized by an unusual steroidal skeleton and have been found to inhibit differentially the proliferation of various mammalian cells in culture by an unknown mechanism. We describe a comprehensive route for the synthesis of cortistatins from a common precursor, which in turn is assembled from two fragments of similar structural complexity. Cortistatins A and J, and for the first time, K and L, have been synthesized in parallel processes from like intermediates prepared from a single compound. With the identification of facile laboratory transformations linking intermediates in the cortistatin L synthetic series with corresponding intermediates to cortistatins A and J, we have been led to speculate that somewhat related paths might occur in nature, offering potential sequencing and chemical detail for cortistatin biosynthetic pathways.

A new and versatile synthesis of substituted isoquinolines: lithiated o-tolualdehyde tert-butylimines are shown to condense with nitriles to form eneamido anion intermediates that can be trapped in situ with various electrophiles, affording a diverse array of highly substituted isoquinolines, many of which are difficult to access by known methods. Further substitutional diversification can be achieved by modification of the work-up conditions and by subsequent transformations. This method should be useful for the preparation of biological active isoquinolines, such as analogs of the isoquinoline-containing natural product cortistatin A.

A hallmark of cancer is unbridled proliferation that can result in increased demand for de novo synthesis of purine and pyrimidine bases required for DNA and RNA biosynthesis. These synthetic pathways are frequently upregulated in cancer and involve various folate-dependent enzymes. Antifolates have a proven record as clinically used oncolytic agents. Our recent research efforts have produced LSN 3213128 (compound 28a), a novel, selective, nonclassical, orally bioavailable antifolate with potent and specific inhibitory activity for aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT), an enzyme in the purine biosynthetic pathway. Inhibition of AICARFT with compound 28a results in dramatic elevation of 5-aminoimidazole 4-carboxamide ribonucleotide (ZMP) and growth inhibition in NCI-H460 and MDA-MB-231met2 cancer cell lines. Treatment with this inhibitor in a murine based xenograft model of triple negative breast cancer (TNBC) resulted in tumor growth inhibition.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

KRAS-G12C is an important oncogenic mutation in patients with NSCLC, CRC, and other cancer types. Currently, there are no FDA-approved KRAS-G12C inhibitors, and those in clinical development have relatively modest activity compared to other approved therapies targeting other classic oncogenic drivers. This modest activity may be potentially due in part to incomplete target occupancy and trapping of mutant KRAS in the inactive GDP-bound state. Achieving maximal clinical benefit in patients harboring a KRAS-G12C mutation, may require a potent inhibitor capable of achieving near complete target engagement. Here, we report the identification of LY3537982, a novel, highly selective and potent inhibitor of the KRAS-G12C protein, discovered using structure-based design. In kinetic studies, LY3537982 showed a high Kinact/Ki value (248,016 M-1 s-1), compared to AMG510 (7,220 M-1 s-1) and MRTX849 (35,000 M-1 s-1). LY3537982 inhibited KRAS-GTP loading with an IC50 value of 3.35 nM in the KRAS-G12C mutant H358 lung cancer cell line, while AMG510 and MRTX849 had IC50 values of 47.9 nM and 89.9 nM, respectively. LY3537982 also inhibited phospho-ERK in H358 cells with an IC50 value of 0.65 nM, while the IC50 values of AMG510 and MRTX849 were 13.5 nM and 14 nM, respectively. In a panel of cancer cell lines with KRAS-G12C or non-G12C mutations, LY3537982 selectively inhibited the growth of KRAS-G12C mutant tumor cells and not KRAS wild-type or non-G12C mutant cells. Sensitivity to LY3537982 varied among the KRAS-G12C mutant cells tested, suggesting that not all cell lines maintain the same dependence on KRAS-G12C. Similarly, in multiple xenograft or patient-derived xenograft (PDX) models harboring a KRAS-G12C mutation, LY3537982 exhibited a range of anti-tumor activity from complete regression to significant tumor growth inhibition, at 3 to 30 mg/kg QD or BID. Mechanism-based combinational screens have also identified certain targeted therapies that can synergize with LY3537982 to achieve better anti-tumor activity in vitro and in vivo, including abemaciclib, the selective AurA inhibitor LY3295668, and cetuximab. Together these data suggest that in certain biologic contexts, broader and more durable anti-tumor activity could be achieved with combination regimens. A first-in-human Phase 1 clinical trial is planned for 2021. Citation Format: Sheng-Bin Peng, Chong Si, Youyan Zhang, Robert D. Van Horn, Xi Lin, Xueqian Gong, Lysiane Huber, Gregory Donoho, Carmen Curtis, John M. Strelow, Wayne P. Bocchinfuso, Deqi Guo, Serge L. Boulet, David Barda, Danalyn Manglicmot, Melbert-Brian D. Saflor, Jing Wang, Junpeng Xiao, Michael J. Chalmers, Lee Burns, Ryan J. Linder, Bradley L. Ackermann, Paul D. Cornwell, Lian Zhou, Denis McCann, James Henry. Preclinical characterization of LY3537982, a novel, highly selective and potent KRAS-G12C inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1259.

In this paper, we report for the first time two enantioselective routes to 4,4-difluoropyrrolidin-3-ol, a valuable building block in medicinal chemistry. In the first route, we took advantage of the C2 symmetry of (3R,4R)-3,4-dihydroxypyrrolidine in which the desired chirality was derived from the chiral pool (l-(+)-tartaric acid). In the second route, we efficiently assembled the pyrrolidine ring in the presence of a gem-difluoro moiety to avoid using potentially hazardous deoxofluorinating reagents and subsequently introduced the chirality by a stereoselective iridium-diamine-catalyzed asymmetric transfer hydrogenation reaction.