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Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1–4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR–Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB–FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Cyclin E1, the protein product of the CCNE1 gene, complexes with CDK2 and is a key regulator of the G1-S transition in cycling cells. CCNE1 amplification has been associated with increased replication stress and genomic instability associated with tumorigenesis. The serine-threonine protein kinase family member PKMYT1 negatively regulates the G2-Mitosis transition by phosphorylating and inactivating CDK1. The aim of the current study was to evaluate the impact of RP-6306, a novel and selective PKMYT1 inhibitor, on the CCNE1 amplified human breast cancer cell line, HCC1569. RP-6306 is a highly potent PKMYT1 inhibitor that displays single digit nM potency in an in vitro enzyme assay. RP-6306 dose-dependently inhibited the phosphorylation of CDK1 on Thr14 in HCC1569 cells and had no impact on the Tyr15 phospho-site of CDK1 that is regulated by family member Wee1. Cell-based assays showed increased phosphorylation of replication stress and pre-mitotic entry biomarkers in RP-6306 treated HCC1569 cells. Micronuclei and Caspase-3 were detected in a dose-dependent manner in HCC1569 cells treated with RP-6306 indicating the onset of genomic instability and apoptosis in these cells. Growth assays confirmed irreparable damage and proliferation defects in cells treated with RP-6306. Experiments to evaluate the combination of RP-6306 with gemcitabine, an S-phase specific pyrimidine analog that inhibits DNA synthesis showed profound synergistic growth defects in HCC1569 cells. In vivo, RP-6306 inhibition of Thr14 phosphorylation of CDK1 in HCC1569 tumors was directly proportional to free circulating plasma levels and resulted in significant inhibition of tumor growth in a dose and time dependent manner. In combination with gemcitabine, RP-6306 demonstrated tumor regression and superior efficacy compared to single agent treatment of either agent alone in multiple CCNE1 amplified models, including HCC1569 and OVCAR3. Our studies show that inhibition of PKMYT1 kinase activity impairs the growth of CCNE1 amplified cancer cell lines both in vitro and in vivo and stands to benefit cancer patients with CCNE1 amplification. A Phase I clinical trial (NCT04855656- Mythic Study) is currently underway to evaluate RP-6306 in patients with advanced solid tumors. Citation Format: Jimmy Fourtounis, John Martino, Rino Stocco, Prasamit Baruah, Nicole Duffy, David Gallo, Sarah Fournier, JingJing Li, Li Li, Elia Aguado, Adam Petrone, Anne Roulston, Yael Mamane, Stephen Morris, Janek Szychowski, Robert Papp, Mike Zinda, C. Gary Marshall. RP-6306, a novel PKMYT1 inhibitor, demonstrates synthetic lethality as monotherapy and in combination with gemcitabine in CCNE1 amplified cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5650.

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, deltaC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.

Background Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system. Methods Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools. Results An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts. Conclusions These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.

We describe a novel method of characterizing protein-RNA interactions using a fluorescence-based multiwell capillary electrophoresis platform based on microfluidic technology. As a proof of concept, we studied the binding of human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) to the transactivation-responsive RNA (TAR). We established conditions to quantify the binding of recombinant HIV-1 Tat to TAR RNA and validated the assay by demonstrating the dependence of this interaction on the presence of the UCU bulge in TAR. In addition, we showed that neomycin inhibited Tat-TAR binding in a dose-dependent manner (IC(50)=1.6-3.0μM). This microfluidic-based method is high-throughput screening compatible and may be applicable to targeting other nucleic acid-protein interactions.

In the brain, DNA fragmentation is associated with apoptotic cell death following ischemic/excitotoxic damage. Fragmented DNA can be detected in situ by labeling the 3'OH termini of the internucleosomal generated fragments with deoxynucleotides, through a process known as terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling, or TUNEL. TUNEL is frequently being used to assess neuronal death following cerebral ischemia in a number of animal models. However, conventional techniques for TUNEL can be time consuming, and are often subjective and thus can lead to inconsistencies among investigators. Moreover, the lack of tools for its quantification and standardization limits the use of this technique in assessing the magnitude of cell death. In the present report, we describe an improved higher throughput technique for TUNEL staining at room temperature on a BioGenex automated stainer, and its subsequent quantitative analysis using NORTHERN ECLIPSE, an imaging analysis program. Its implementation allows us to effectively quantify TUNEL positive cells in the CA1 region of the hippocampus following global forebrain ischemia in rats. We conclude that this general histological technique can be applied to the study of cell death in numerous other experimental models.