Generation of two human induced pluripotent stem cell lines with BAX and BAK1 double knock-out using CRISPR/Cas9.

Bcl-2-associated X protein (BAX) and Blc-2 homologous antagonist killer 1 (BAK) are two pro-apoptotic members of BCL2 family. Here, two BAX/BAK double knock-out human induced pluripotent stem cell lines (iPSC) we generated using CRISPR-Cas9 to generate apoptosis incompetent cell lines. The resulting cell lines were karyotypically normal, had typical morphology and expressed typical markers for the undifferentiated state.

Generation of THRB-GS(E125G_G126S) and THRB-KO human iPSC lines to study noncanonical thyroid hormone signalling.

THRB is a nuclear receptor, regulating gene expression dependent on thyroid hormone (TH) binding. The same receptor mediates signaling pathway activation in the cytosol. The challenge is to distinguish which of the two mechanisms is responsible for physiological effects of TH. We established an iPSC cell line with two mutations (E125G_G126S) in the THRB DNA-binding domain, which abrogates nuclear action and, thus, allows to study signaling pathway activation exclusively. We also generated a THRB knockout cell line to abolish all THRB effects. Comparison of WT and these two cell lines allows attribution of thyroid hormone effects to the underlying mechanism.

Generation of iPSC lines with SLC16A2:G401R or SLC16A2 knock out.

The X-linked Allan-Herndon-Dudley syndrome (AHDS) is characterized by severely impaired psychomotor development and is caused by mutations in the SLC16A2 gene encoding the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). By targeting exon 3 of SLC16A2 using CRISPR/Cas9 with single-stranded oligodeoxynucleotides as homology-directed repair templates, we introduced the AHDS patient missense variant G401R and a novel knock-out deletion variant (F400Sfs*17) into the male healthy donor hiPSC line BIHi001-B. We successfully generated cerebral organoids from these genome-edited lines, demonstrating the utility of the novel lines for modelling the effects of MCT8-deficency on human neurodevelopment.

Identifying the effectiveness of 3D culture systems to recapitulate breast tumor tissue in situ.

PURPOSE: Breast cancer heterogeneity contributes to chemotherapy resistance and decreased patient survival. To improve patient outcomes it is essential to develop a technology that is able to rapidly select the most efficacious therapy that targets the diverse phenotypes present within the tumor. Breast cancer organoid technologies are proposed as an attractive approach for evaluating drug responses prior to patient therapy. However, there remain challenges in evaluating the effectiveness of organoid cultures to recapitulate the heterogeneity present in the patient tumor in situ. METHOD: Organoids were generated from seven normal breast and nineteen breast cancer tissues diagnosed as estrogen receptor positive or triple negative. The Jensen-Shannon divergence index, a measure of the similarity between distributions, was used to compare and evaluate heterogeneity in starting tissue and their resultant organoids. Heterogeneity was analyzed using cytokeratin 8 and cytokeratin 14, which provided an easily scored readout. RESULTS: In the in vitro culture system HER1 and FGFR were able to drive intra-tumor heterogeneity to generate divergent phenotypes that have different sensitivities to chemotherapies. CONCLUSION: Our methodology, which focuses on quantifiable cellular phenotypes, provides a tractable system that complements omics approaches to provide an unprecedented view of heterogeneity and will enhance the identification of novel therapies and facilitate personalized medicine.

ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs.

CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening.

The key features of SARS-CoV-2 leader and NSP1 required for viral escape of NSP1-mediated repression.

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in the viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19, and 20) and another within NSP1 (R124), which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the antisense oligo (ASO) to efficiently and specifically down-regulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of antiviral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

FACS protocol for direct comparison of cell populations isolated from mice.

A FACS protocol is described that eliminates isolation and staining artifacts to allow accurate comparison between cell populations isolated from organs obtained from disparate mouse groups. This protocol was validated by characterizing the estrogen receptor positive cells within the mammary gland of transgenic mice with different genotypes at different stages of the estrous cycle. We include protocols necessary to batch stage animals within the cycle to proceed directly to FACS, which provides optimal RNA yields for RNA-seq. For complete details on the use and execution of this protocol, please refer to Ludwik et al. (2020).

RSK2 and ERα comrades-in-arms in homeostasis and transformation.

The physiological response to estrogen differs according to the developmental stage. We show, in the adult, estrogen-responsiveness is driven by ERK1/2 (extracellular signal-regulated kinase 1/2) whereas its downstream effector, RSK2 (p90 ribosomal S6 kinase 2), prevents continuous ERK1/2 activity through regulation of oxidative stress. Bioinformatic analysis revealed RSK2 association with breast cancer risk and oral contraceptives.

RSK2 Maintains Adult Estrogen Homeostasis by Inhibiting ERK1/2-Mediated Degradation of Estrogen Receptor Alpha.

In response to estrogens, estrogen receptor alpha (ERα), a critical regulator of homeostasis, is degraded through the 26S proteasome. However, despite the continued presence of estrogen before menopause, ERα protein levels are maintained. We discovered that ERK1/2-RSK2 activity oscillates during the estrous cycle. In response to high estrogen levels, ERK1/2 is activated and phosphorylates ERα to drive ERα degradation and estrogen-responsive gene expression. Reduction of estrogen levels results in ERK1/2 deactivation. RSK2 maintains redox homeostasis, which prevents sustained ERK1/2 activation. In juveniles, ERK1/2-RSK2 activity is not required. Mammary gland regeneration demonstrates that ERK1/2-RSK2 regulation of ERα is intrinsic to the epithelium. Reduced RSK2 and enrichment in an estrogen-regulated gene signature occur in individuals taking oral contraceptives. RSK2 loss enhances DNA damage, which may account for the elevated breast cancer risk with the use of exogenous estrogens. These findings implicate RSK2 as a critical component for the preservation of estrogen homeostasis.

Genome-wide analysis of RNA and protein localization and local translation in mESC-derived neurons.

The subcellular localization and translation of mRNAs are fundamental biological processes. In neurons, they underlie cell growth and synaptic plasticity, which serves as a foundation of learning and memory. Multiple approaches have been developed to separate neurons on subcellular compartments - cell bodies (soma) and cell extensions (axons and dendrites) - for further biochemical analyses. Here we describe neurite/soma separation approach in combination with RNA sequencing and proteomic analyses to identify localized and locally translated RNAs and proteins. This approach allows quantification of around 7000 of local proteins and the entire local transcriptome. It provides a powerful tool for investigation of the mechanisms underlying RNA localization and local translation in neurons.

ERα-Mediated Nuclear Sequestration of RSK2 Is Required for ER+ Breast Cancer Tumorigenesis.

Although ribosomal protein S6 kinase A3 (RSK2) activation status positively correlates with patient responses to antiestrogen hormonal therapies, the mechanistic basis for these observations is unknown. Using multiple in vitro and in vivo models of estrogen receptor-positive (ER+) breast cancer, we report that ERα sequesters active RSK2 into the nucleus to promote neoplastic transformation and facilitate metastatic tumor growth. RSK2 physically interacted with ERα through its N terminus to activate a proneoplastic transcriptional network critical to the ER+ lineage in the mammary gland, thereby providing a gene signature that effectively stratified patient tumors according to ERα status. ER+ tumor growth was strongly dependent on nuclear RSK2, and transgenic mice engineered to stably express nuclear RSK2 in the mammary gland developed high-grade ductal carcinoma in situ Mammary cells isolated from the transgenic model and introduced systemically successfully disseminated and established metastatic lesions. Antiestrogens disrupted the interaction between RSK2 and ERα, driving RSK2 into the cytoplasm and impairing tumor formation. These findings establish RSK2 as an obligate participant of ERα-mediated transcriptional programs, tumorigenesis, and divergent patient responses to antiestrogen therapies.Significance: Nuclear accumulation of active RSK drives a protumorigenic transcriptional program and renders ER+ breast cancer susceptible to endocrine-based therapies. Cancer Res; 78(8); 2014-25. ©2018 AACR.

Stereoselective Synthesis and Evaluation of C6″-Substituted 5a-Carbasugar Analogues of SL0101 as Inhibitors of RSK1/2.

A convergent synthesis of 5a-carbasugar analogues of the n-Pr-variant of SL0101 is described. The analogues were synthesized in an effort to find compounds with potent in vivo efficacy in the inhibition of p90 ribosomal s6 kinase (RSK1/2). The synthesis derived the desired C-4 L-rhamnose stereochemistry from quinic acid and used a highly selective cuprate addition, NaBH4 reduction, Mitsunobu inversion, and alkene dihydroxylation to install the remaining stereochemistry. A Pd-catalyzed cyclitolization stereoselectively installed the aglycon at the anomeric position. The analogues were evaluated as RSK1/2 inhibitors and found to have 3- to 6-fold improved activity.

Development of a RSK Inhibitor as a Novel Therapy for Triple-Negative Breast Cancer.

Metastatic breast cancer is an incurable disease and identification of novel therapeutic opportunities is vital. Triple-negative breast cancer (TNBC) frequently metastasizes and high levels of activated p90RSK (RSK), a downstream MEK-ERK1/2 effector, are found in TNBC. We demonstrate, using direct pharmacologic and genetic inhibition of RSK1/2, that these kinases contribute to the TNBC metastatic process in vivo Kinase profiling showed that RSK1 and RSK2 are the predominant kinases targeted by the new inhibitor, which is based on the natural product SL0101. Further evidence for selectivity was provided by the observations that silencing RSK1 and RSK2 eliminated the ability of the analogue to further inhibit survival or proliferation of a TNBC cell line. In vivo, the new derivative was as effective as the FDA-approved MEK inhibitor trametinib in reducing the establishment of metastatic foci. Importantly, inhibition of RSK1/2 did not result in activation of AKT, which is known to limit the efficacy of MEK inhibitors in the clinic. Our results demonstrate that RSK is a major contributor to the TNBC metastatic program and provide preclinical proof-of-concept for the efficacy of the novel SL0101 analogue in vivo Mol Cancer Ther; 15(11); 2598-608. ©2016 AACR.

Ribosomal S6 kinase (RSK) modulators: a patent review.

INTRODUCTION: The p90 ribosomal S6 kinases (RSK) are a family of Ser/Thr protein kinases that are downstream effectors of MEK1/2-ERK1/2. Increased RSK activation is implicated in the etiology of multiple pathologies, including numerous types of cancers, cardiovascular disease, liver and lung fibrosis, and infections. AREAS COVERED: The review summarizes the patent and scientific literature on small molecule modulators of RSK and their potential use as therapeutics. The patents were identified using World Intellectual Property Organization and United States Patent and Trademark Office databases. The compounds described are predominantly RSK inhibitors, but a RSK activator is also described. The majority of the inhibitors are not RSK-specific. EXPERT OPINION: Based on the overwhelming evidence that RSK is involved in a number of diseases that have high mortalities it seems surprising that there are no RSK modulators that have pharmacokinetic properties suitable for in vivo use. MEK1/2 inhibitors are in the clinic, but the efficacy of these compounds appears to be limited by their side effects. We hypothesize that targeting the downstream effectors of MEK1/2, like RSK, are an untapped source of drug targets and that they will generate less side effects than MEK1/2 inhibitors because they regulate fewer effectors.