Because of their somatic cell origin, human induced pluripotent stem cells (HiPSCs) are assumed to carry a normal diploid genome, and adaptive chromosomal aberrations have not been fully evaluated. Here, we analyzed the chromosomal integrity of 66 HiPSC and 38 human embryonic stem cell (HESC) samples from 18 different studies by global gene expression meta-analysis. We report identification of a substantial number of cell lines carrying full and partial chromosomal aberrations, half of which were validated at the DNA level. Several aberrations resulted from culture adaptation, and others are suspected to originate from the parent somatic cell. Our classification revealed a third type of aneuploidy already evident in early passage HiPSCs, suggesting considerable selective pressure during the reprogramming process. The analysis indicated high incidence of chromosome 12 duplications, resulting in significant enrichment for cell cycle-related genes. Such aneuploidy may limit the differentiation capacity and increase the tumorigenicity of HiPSCs.

Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.

The unique abilities of human pluripotent stem cells to self-renew and to differentiate into cells of the three germ layers make them an invaluable tool for the future of regenerative medicine. However, the same properties also make them tumorigenic, and therefore hinder their clinical application. Hence, the tumorigenicity of human embryonic stem cells (HESCs) has been extensively studied. Until recently, it was assumed that human induced pluripotent stem cells (HiPSCs) would behave like their embryonic counterparts in respect to their tumorigenicity. However, a rapidly accumulating body of evidence suggests that there are important genetic and epigenetic differences between these two cell types, which seem to influence their tumorigenicity.

The CRISPR-Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy number gain, CRISPR-Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell cycle arrest. By examining single guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR-Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this technology for identification of essential genes in amplified regions.

Patient-derived xenografts (PDXs) have become a prominent cancer model system, as they are presumed to faithfully represent the genomic features of primary tumors. Here we monitored the dynamics of copy number alterations (CNAs) in 1,110 PDX samples across 24 cancer types. We observed rapid accumulation of CNAs during PDX passaging, often due to selection of pre-existing minor clones. CNA acquisition in PDXs was correlated with the tissue-specific levels of aneuploidy and genetic heterogeneity observed in primary tumors. However, the particular CNAs acquired during PDX passaging differed from those acquired during tumor evolution in patients. Several CNAs recurrently observed in primary tumors gradually disappeared in PDXs, indicating that events undergoing positive selection in humans can become dispensable during propagation in mice. Importantly, the genomic stability of PDXs was associated with their response to chemotherapy and targeted drugs. These findings have important implications for PDX-based modeling of human cancer.

Anticancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth-inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM (profiling relative inhibition simultaneously in mixtures), a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the molecular features of the cell lines. Our findings include compounds that killed by inducing phosphodiesterase 3A-Schlafen 12 complex formation, vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2, the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins, and the anti-inflammatory drug tepoxalin, which killed via the multidrug resistance protein ATP-binding cassette subfamily B member 1 (ABCB1). The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation. NATURE CANCER | VOL 1 | FeBRUARY 2020 | 235-248 | www.nature.com/natcancer 235 ResouRce NATuRE CANCER the remaining compounds being either chemotherapeutics (2%) or targeted oncology agents (21%).Screening results. We employed a 2-stage screening strategy whereby drugs were first screened in triplicate at a single dose (2.5 µM); 1,448 drugs screening positives were then rescreened in triplicate in an eight-point dose-response ranging from 10 µM to 610 pM ( Fig. 1c and Supplementary Table 2). Interestingly, most active compounds (774 out of 1,448, 53%) were originally developed for non-oncology clinical indications (Fig. 1d). The primary and secondary screening datasets are available on the Cancer Dependency Map portal (https://depmap.org/repurposing) and figshare (https://doi.org/10.6084/m9.figshare.9393293; Extended Data Figs. 1-4). We compared the PRISM results to two gold standard datasets: GDSC (ref. 2 ) and CTD 2 (ref. 3 ). The three datasets shared 84 compounds tested on a median of 236 common cell lines, yielding 16,650 shared data points. The PRISM dataset had a similar degree of concordance to GDSC and CTD 2 (Pearson correlations of 0.60 and 0.61, respectively over all shared data points), as the GDSC and CTD 2 datasets had to each other (Pearson correlation 0.62) (Extended Data Fig. 5a). The three datasets remained similarly concordant when the analysis was restricted to data points showing evidence of anticancer activity (Extended Data Fig. 5b). We conclude that, despite differences in assay format, sources of compounds 5 and sources of cell lines 6 , the PRISM Repurposing dataset is similarly robust compared to existing pharmacogenomic datasets.At the level of individual compound dose-responses, we note that the PRISM Repurposing dataset tends to be somewhat noisier, with a higher standard error estimated from vehicle contr...

Cancer is driven by multiple types of genetic alterations, which range in size from point mutations to whole chromosome gains and losses, a condition known as aneuploidy.Chromosome instability, the process that gives rise to aneuploidy, can promote tumorigenesis by increasing genetic heterogeneity and promoting tumor evolution. However, much less is known about how aneuploidy itself contributes to tumor formation and progression. Unlike some pancancer oncogenes and tumor suppressor genes that drive transformation in virtually all cell types and cellular contexts, aneuploidy is not a universal promoter of tumorigenesis. Instead, a picture emerges that paints aneuploidy as a context-dependent cancer type-specific oncogenic event. In this Review, we discuss the role of aneuploidy in tumor development, and its clinical relevance as a prognostic marker and as a potential therapeutic target.

The use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns), nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn #1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs. PluriSIn #1 was also cytotoxic to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state. Finally, application of PluriSIn #1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments.