Rational design of next-generation therapeutics can be facilitated by high-resolution structures of drug targets bound to small-molecule inhibitors. However, application of structure-based methods to macromolecules refractory to crystallisation has been hampered by the often-limiting resolution and throughput of cryogenic electron microscopy (cryo-EM). Here, we use high-resolution cryo-EM to determine structures of the CDK-activating kinase, a master regulator of cell growth and division, in its free and nucleotide-bound states and in complex with 14 inhibitors at up to 1.8 Å resolution. Our structures provide detailed insight into inhibitor interactions and networks of water molecules in the active site of cyclin-dependent kinase 7. Our data support a previously proposed mechanism contributing to inhibitor selectivity, thereby providing the basis for rational design of next-generation therapeutics. Additionally, our results establish a methodological framework for the use of high-resolution cryo-EM in structure-based drug design.
b-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. However, little is known on the genetic and epigenetic changes linked with the dedifferentiation of b-cells. We now report that b-cell dedifferentiation is associated with epithelial to mesenchymal transition (EMT) triggered by miR-7-mediated repression of Smarca4/Brg1 expression, a catalytic subunit of the mSwi/Snf chromatin remodeling complexes essential for b-cell transcription factors (b-TFs) activity. miR-7-mediated repression of Brg1 expression in diabetes causes an overall compaction of chromatin structure preventing b-TFs from accessing and transactivating genes maintaining the functional and epithelial identity of b-cells. Concomitantly, loss of b-cell identity impairs the ability of b-TFs Pdx1, Nkx6-1, Neurod1 to repress non-b-cell genes enriched selectively in mesenchymal cells leading to EMT, change in islet microenvironment, and fibrosis. Remarkably, anti-EMT agents normalized glucose tolerance of diabetic mice, thus revealing mesenchymal reprogramming of b-cells as a novel therapeutic target in diabetes. This study sheds light on the genetic signature of dedifferentiated b-cells and highlights how loss of mSwi/Snf activity in diabetes initiating a step-wise remodeling of epigenetic landscapes of b-cells leading to the induction of an EMT process reminiscent of a response to tissue injury. specific miR-7 overexpressing mouse model (Tg7) which develops early-onset diabetes due to b-cell dedifferentiation (32). Conversely, how elevated miR-7 levels in diabetes impair b-cell identity remains unclear. By profiling gene expression and open chromatin regions of Tg7 islets, we now report that loss of b-cell identity triggers an islet injury response characterized by an epithelial to mesenchymal transition (EMT) process in both mouse and humans. Elevated miR-7 levels trigger the dedifferentiation of mature b-cells through the repression of Brg1/Smarca4, a catalytic subunit of mSwi/Snf chromatin remodelling complexes required for the transactivation lineagespecific and epithelial genes by b-cell transcription factors (b-TF) function. Our findings highlight how dysregulation miRNA networks can compromise the functional and epithelial identity of b-cells and trigger an EMT-like reprogramming process with therapeutic relevance in diabetes. RESULTS Loss of b-cells identity impairs islet connectivity in Tg7 miceGenetic overexpression of miR-7 in b-cells results in diabetes from 4 weeks of age and is associated with decreased expression of mature b-cell differentiation markers, most notably INS1/2 and b-TFs genes ( Supplementary Figure 1). How dedifferentiation affects Ca 2+ dynamics and long-range b-cell:b-cell connectivity in diabetes remains unclear. To this end, we monitored intracellular free Ca 2+ dynamics in intact islets in response to elevated concentrations of glucose using a trappable intracellular Ca 2+ dye and multicellular imaging.Ca 2+ dynamics in response to 17 mM glucose were almost...
Breast cancer (BC) is the most prevalent cancer in women worldwide. Despite initial sensitivity to endocrine therapies in hormone receptor positive BC, high mortality is driven by treatment-resistant disease. While several highly effective targeted treatments have been developed, resistance is common in the metastatic setting, highlighting the need for new therapies. Cyclin-dependent kinases (CDKs) have emerged as important targets for cancer treatment due to their integral role in driving the cell cycle. CDK4/6 inhibitors have advanced strongly in ER+ BC and are now becoming standard-of-care. However, resistance is a major problem. To address this, we have targeted CDK7, a master regulator of the cell cycle CDKs and transcription processes, both commonly dysregulated in cancer. CDK7 inhibition selectively inhibits cancer cell growth due to their reliance on aberrant gene expression and/or unregulated cell cycle progression. ICEC0942 (Samuraciclib), discovered in our lab at Imperial College London, has recently progressed to Phase 2 clinical trials. Considering the current clinical development of this and other CDK7 inhibitors (CDK7i), it is important to determine which patients will best respond to Samuraciclib. Towards identifying modulators of response to Samuraciclib, we performed a genome-wide CRISPR/Cas9-knockout screen in MCF7 cells. The mTOR/PI3K pathway was identified as important for response to CDK7i. Knockout of mTOR activators reduced sensitivity to Samuraciclib, while knockout of mTOR repressors increased sensitivity. Next, we studied the role of mutations in the catalytic subunit of PI3K (PIK3CA), which occur in nearly 30% of BC cases, in response to Samuraciclib. We utilized an isogenic panel of MCF10A cell lines, which were either wild-type for PIK3CA or had a heterozygous knock-in of either of the two most common mutations (E545K or H1047R). Cell growth was monitored following long-term (42-day) treatment with Samuraciclib. The drug was washed out (day 15) to determine the role of PIK3CA mutations in drug-induced growth arrest. We find that although Samuraciclib effectively inhibits growth of all cell lines, regardless of PIK3CA mutation status, the longevity of growth arrest post-washout is promoted by activating PIK3CA mutations. Finally, we assessed the role of mTOR and its downstream effectors in Samuraciclib response. Cell proliferation of MCF7 cells treated with Samuraciclib or in combination with mTOR/PI3K/AKT inhibitors was measured via flow cytometry. Samuraciclib treatment arrested cell proliferation; however, proliferation was reverted in combination with an mTOR/PI3K/AKT inhibitor. In conclusion, our findings highlight the importance of an active mTOR/PI3K pathway in promoting response to Samuraciclib. This may help guide patient treatments and development of effective combination therapies. Citation Format: Kaste Jurgaityte, Georgina Sava, Chun Fui Lai, Hailing Fan, Van Nguyen, Laki Buluwela, Simak Ali. Defining modulators of response to samuraciclib, a CDK7 inhibitor for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1557.
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