SummaryNeurofibromatosis type 1 (NF1) is a tumor predisposition genetic disease caused by mutations in the NF1 tumor suppressor gene. Plexiform neurofibromas (PNFs) are benign Schwann cell (SC) tumors of the peripheral nerve sheath that develop through NF1 inactivation and can progress toward a malignant soft tissue sarcoma. There is a lack of non-perishable model systems to investigate PNF development. We reprogrammed PNF-derived NF1(−/−) cells, descendants from the tumor originating cell. These NF1(−/−)-induced pluripotent stem cells (iPSCs) captured the genomic status of PNFs and were able to differentiate toward neural crest stem cells and further to SCs. iPSC-derived NF1(−/−) SCs exhibited a continuous high proliferation rate, poor myelination ability, and a tendency to form 3D spheres that expressed the same markers as their PNF-derived primary SC counterparts. They represent a valuable model to study and treat PNFs. PNF-derived iPSC lines were banked for making them available.

The spatial partitioning of the transcriptome in the cell is an important form of gene-expression regulation. Here, we address how intron retention influences the spatio-temporal dynamics of transcripts from two clinically relevant genes: TERT (Telomerase Reverse Transcriptase) pre-mRNA and TUG1 (Taurine-Upregulated Gene 1) lncRNA. Single molecule RNA FISH reveals that nuclear TERT transcripts uniformly and robustly retain specific introns. Our data suggest that the splicing of TERT retained introns occurs during mitosis. In contrast, TUG1 has a bimodal distribution of fully spliced cytoplasmic and intron-retained nuclear transcripts. We further test the functionality of intron-retention events using RNA-targeting thiomorpholino antisense oligonucleotides to block intron excision. We show that intron retention is the driving force for the nuclear compartmentalization of these RNAs. For both RNAs, altering this splicing-driven subcellular distribution has significant effects on cell viability. Together, these findings show that stable retention of specific introns can orchestrate spatial compartmentalization of these RNAs within the cell. This process reveals that modulating RNA localization via targeted intron retention can be utilized for RNA-based therapies.

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Primate-specific NBL2 macrosatellite is hypomethylated in several types of tumors, yet the consequences of this DNA hypomethylation remain unknown. We show that NBL2 conserved repeats are close to the centromeres of most acrocentric chromosomes. NBL2 associates with the perinucleolar region and undergoes severe demethylation in a subset of colorectal cancer (CRC). Upon DNA hypomethylation and histone acetylation, NBL2 repeats are transcribed in tumor cell lines and primary CRCs. NBL2 monomers exhibit promoter activity, and are contained within novel, non-polyA antisense lncRNAs, which we designated TNBL (Tumor-associated NBL2 transcript). TNBL is stable throughout the mitotic cycle, and in interphase nuclei preferentially forms a perinucleolar aggregate in the proximity of a subset of NBL2 loci. TNBL aggregates interact with the SAM68 perinucleolar body in a mirror-image cancer specific perinucleolar structure. TNBL binds with high affinity to several proteins involved in nuclear functions and RNA metabolism, such as CELF1 and NPM1. Our data unveil novel DNA and RNA structural features of a non-coding macrosatellite frequently altered in cancer.

Analysis of cancer mutagenic signatures provides information about the origin of mutations and can inform the use of clinical therapies, including immunotherapy. In particular, APOBEC3A (A3A) has emerged as a major driver of mutagenesis in cancer cells, and its expression results in DNA damage and susceptibility to treatment with inhibitors of the ATR and CHK1 checkpoint kinases. Here, we report the implementation of CRISPR/Cas-9 genetic screening to identify susceptibilities of multiple A3A-expressing lung adenocarcinoma (LUAD) cell lines. We identify HMCES, a protein recently linked to the protection of abasic sites, as a central protein for the tolerance of A3A expression. HMCES depletion results in synthetic lethality with A3A expression preferentially in a TP53-mutant background. Analysis of previous screening data reveals a strong association between A3A mutational signatures and sensitivity to HMCES loss and indicates that HMCES is specialized in protecting against a narrow spectrum of DNA damaging agents in addition to A3A. We experimentally show that both HMCES disruption and A3A expression increase susceptibility of cancer cells to ionizing radiation (IR), oxidative stress, and ATR inhibition, strategies that are often applied in tumor therapies. Overall, our results suggest that HMCES is an attractive target for selective treatment of A3A-expressing tumors.

CRISPR/Cas9 gene editing can inactivate genes in a precise manner. This process involves DNA double-strand breaks (DSB), which may incur a loss of cell fitness. We hypothesize that DSB toxicity may be variable depending on the chromatin environment in the targeted locus. Here, by analyzing isogenic cell line pair CRISPR experiments jointly with previous screening data from across ~900 cell lines, we show that TP53-associated break toxicity is higher in genomic regions that harbor active chromatin, such as gene regulatory elements or transcription elongation histone marks. DSB repair pathway choice and DNA sequence context also associate with toxicity. We also show that, due to noise introduced by differential toxicity of sgRNA-targeted sites, the power of genetic screens to detect conditional essentiality is reduced in TP53 wild-type cells. Understanding the determinants of Cas9 cut toxicity will help improve design of CRISPR reagents to avoid incidental selection of TP53-deficient and/or DNA repair deficient cells.

Background Malignant peripheral nerve sheath tumor (MPNST) constitutes the leading cause of neurofibromatosis type 1–related mortality. MPNSTs contain highly rearranged hyperploid genomes and exhibit a high division rate and aggressiveness. We have studied in vitro whether the mitotic kinesins KIF11, KIF15, and KIF23 have a functional role in maintaining MPNST cell survival and can represent potential therapeutic vulnerabilities. Methods We studied the expression of kinesin mRNAs and proteins in tumors and cell lines and used several in vitro functional assays to analyze the impact of kinesin genetic suppression (KIF15, KIF23) and drug inhibition (KIF11) in MPNST cells. We also performed in vitro combined treatments targeting KIF11 together with other described MPNST targets. Results The studied kinesins were overexpressed in MPNST samples. KIF15 and KIF23 were required for the survival of MPNST cell lines, which were also more sensitive than benign control fibroblasts to the KIF11 inhibitors ispinesib and ARRY-520. Co-targeting KIF11 and BRD4 with ARRY-520 and JQ1 reduced MPNST cell viability, synergistically killing a much higher proportion of MPNST cells than control fibroblasts. In addition, genetic suppression of KIF15 conferred an increased sensitivity to KIF11 inhibitors alone or in combination with JQ1. Conclusions The mitotic spindle kinesins KIF11 and KIF15 and the cytokinetic kinesin KIF23 play a clear role in maintaining MPNST cell survival and may represent potential therapeutic vulnerabilities. Although further in vivo evidences are still mandatory, we propose a simultaneous suppression of KIF11, KIF15, and BRD4 as a potential therapy for MPNSTs.

The study of somatic genetic alterations in tumors contributes to the understanding and management of cancer. Genetic alterations, such us copy number or copy neutral changes, generate allelic imbalances (AIs) that can be determined using polymorphic markers. Here we report the development of a simple set of calculations for analyzing microsatellite multiplex PCR data from control-tumor pairs that allows us to obtain accurate information not only regarding the AI status of tumors, but also the percentage of tumor-infiltrating normal cells, the locus copy-number status and the mechanism involved in AI. We validated this new approach by re-analyzing a set of Neurofibromatosis type 1-associated dermal neurofibromas and comparing newly generated data with results obtained for the same tumors in a previous study using MLPA, Paralog Ratio Analysis and SNP-array techniques.Microsatellite multiplex PCR analysis (MMPA) should be particularly useful for analyzing specific regions of the genome containing tumor suppressor genes and also for determining the percentage of infiltrating normal cells within tumors allowing them to be sorted before they are analyzed by more expensive techniques.