Reprogramming enriches for somatic cell clones with small-scale mutations in cancer-associated genes.

Cellular therapies based on induced pluripotent stem cells (iPSCs) come out of age and an increasing number of clinical trials applying iPSC-based transplants are ongoing or in preparation. Recent studies, however, demonstrated a high number of small-scale mutations in iPSCs. Although the mutational load in iPSCs seems to be largely derived from their parental cells, it is still unknown whether reprogramming may enrich for individual mutations that could lead to loss of functionality and tumor formation from iPSC derivatives. 30 hiPSC lines were analyzed by whole exome sequencing. High accuracy amplicon sequencing showed that all analyzed small-scale variants pre-existed in their parental cells and that individual mutations present in small subpopulations of parental cells become enriched among hiPSC clones during reprogramming. Among those, putatively actionable driver mutations affect genes related to cell-cycle control, cell death, and pluripotency and may confer a selective advantage during reprogramming. Finally, a short hairpin RNA (shRNA)-based experimental approach was applied to provide additional evidence for the individual impact of such genes on the reprogramming efficiency. In conclusion, we show that enriched mutations in curated onco- and tumor suppressor genes may account for an increased tumor risk and impact the clinical value of patient-derived hiPSCs.

Effective drug treatment identified by in vivo screening in a transplantable patient-derived xenograft model of chronic myelomonocytic leukemia.

To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma 1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.

Pooled Generation of Lentiviral Tetracycline-Regulated microRNA Embedded Short Hairpin RNA Libraries.

Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery. Starting from a gene list to the identification of hits, the protocol enables shRNA screens within 6 weeks.

Refined sgRNA efficacy prediction improves large- and small-scale CRISPR-Cas9 applications.

Genome editing with the CRISPR-Cas9 system has enabled unprecedented efficacy for reverse genetics and gene correction approaches. While off-target effects have been successfully tackled, the effort to eliminate variability in sgRNA efficacies-which affect experimental sensitivity-is in its infancy. To address this issue, studies have analyzed the molecular features of highly active sgRNAs, but independent cross-validation is lacking. Utilizing fluorescent reporter knock-out assays with verification at selected endogenous loci, we experimentally quantified the target efficacies of 430 sgRNAs. Based on this dataset we tested the predictive value of five recently-established prediction algorithms. Our analysis revealed a moderate correlation (r = 0.04 to r = 0.20) between the predicted and measured activity of the sgRNAs, and modest concordance between the different algorithms. We uncovered a strong PAM-distal GC-content-dependent activity, which enabled the exclusion of inactive sgRNAs. By deriving nine additional predictive features we generated a linear model-based discrete system for the efficient selection (r = 0.4) of effective sgRNAs (CRISPRater). We proved our algorithms' efficacy on small and large external datasets, and provide a versatile combined on- and off-target sgRNA scanning platform. Altogether, our study highlights current issues and efforts in sgRNA efficacy prediction, and provides an easily-applicable discrete system for selecting efficient sgRNAs.

The non-coding RNA landscape of human hematopoiesis and leukemia.

Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs-such as LINC00173 in granulocytes-and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.While micro-RNAs are known regulators of haematopoiesis and leukemogenesis, the role of long non-coding RNAs is less clear. Here the authors provide a non-coding RNA expression landscape of the human hematopoietic system, highlighting their role in the formation and maintenance of the human blood hierarchy.

An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.

RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system.

Paced right bundle branch block: identification and management of a malpositioned left ventricular pacemaker lead.

Malpositioning of a permanent pacemaker lead in the left ventricle is rare. Usually, a paced right bundle branch pattern is the initial finding that fosters other confirmatory studies such as chest films and transthoracic echocardiogram. We describe the unusual case of an asymptomatic 83-year-old male patient who was incidentally found with a permanent pacemaker lead placed through the atrial septum into the left ventricle. This patient had contraindications for chronic anticoagulation and was placed on antiplatelet therapy instead. He has been well after three years without evidence of embolic episodes.