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Supplementary Table S1 lists biochemical and cellular activity of BRG1/BRM inhibitors. Supplementary Table S2 lists BRM011 and BRM014 activity across cell line panel. Supplementary Table S3 lists cell lines generated for this study. Supplementary Table S4 lists antibody and qPCR probes used in this study.

Annotations for the sgRNA libraries containing sequences, target gene information, Zscore and RNAseq data for each cell line screened.

Supplementary Figure S1 shows genetic dependency profiles for BRAF, MAPK1 (ERK2), and MAPK3 (ERK1) by BRAFV600 mutation status from three independent screens: DRIVE, ACHILLES, and AVANA.

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF–MEK–ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation–induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation.Implications:This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.

All supplementary figure and table titles and legends.

Supplementary Figure Legends, Table Legends

Additional method details for ATAC-Seq Data Analysis and ChIP-qPCR

Figure S1. Cellular phenotypes observed in response to ERK2 overexpression in A-375 cells. Figure S2. No paracrine effect was produced by cells dying due to knockdown of the essential gene PSMA3. Figure S3. A-375 xenograft tumors with ERK2 overexpression displayed necrosis and/or fibrosis. Figure S4. Paradoxical activation triggered by low doses of BRAFi in GR-M augmented the growth suppression caused by ERK2 overexpression. Figure S5. Graphical representations of vector constructs used in this study. Table S1. MSigDB Hallmark pathways that were significantly enriched in genes upregulated after 20 h treatment with doxycycline in A-375 ERK2 cells. Table S2. Transcription factor targets (from MSigDB C3 TFT) for known ERK targets were significantly enriched in genes upregulated after 20 h treatment with doxycycline in A-375 ERK2 cells.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues.Implications:BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

Supplementary Figure S1. Comparison of drop out phenotypes in MKN45, RKO, HT1080 highlighting selected pan-lethal genes. Supplementary Figure S2. The genes that scored as lethal by both RNAi and CRISPR were strongly enriched for known essential genes classes. Supplementary Figure S3. To identify likely off-target hits the lethality scores of non-expressed genes were examined, as they are expected not to be required for cell viability. Supplementary Figure S4. shRNAs directed towards CDK9 do not show robust protein depletion. Supplementary Figure S5. Additional methods measuring the proliferation effects of individual sgRNA/shRNAs to validate the impact that targeting selected genetic dependencies have on cell viability. Supplementary Figure S6. Correlation analysis displaying features that correlated most significantly with sgRNA potency. Supplementary Figure S7. Effect of relative position within a gene on sgRNA viability effects. Supplementary Figure S8. Non-scoring sgRNA in conserved Pfam domains have a reduced editing efficiency compared to guides with strong viability effects. Supplementary Figure S9. Multiple genomic cuts result in DNA damage induced G2/M cell cycle arrest. Supplementary Figure S10. Multiple genomic cuts lead to an increase in cell death. Supplementary Figure S11. Pie chart demonstrating that the overall contribution of copy number effects in determining essential genes in aneuploid lines is relatively minor.

Supplementary Figure S1 shows additional shRNA knockdown data in uveal melanoma cell lines. Supplementary Figure S2 shows additional dual BRG1/BRM knockdown data and BRG1 rescue data. Supplementary Figure S3 shows basal SWI/SNF subunit expression, additional caspase activity and viability data in compound treated and shRNA knockdown cell lines, and SWI/SNF mutations in uveal melanoma cells lines. Supplementary Figure S4 shows additional MITF knockdown data and further RNA-Seq and ATAC-Seq data set analyses. Supplementary Figure S5 shows target gene modulation by compound treatment in uveal melanoma cell lines and additional MITF overexpression rescue data. Supplementary Figure S6 shows genomic location of primers used in ChIP-qPCR experiment.

Supplementary information containing six supplementary figures (Figure S1-S6), one supplementary table (Table S1) and supplementary materials and methods. Supplementary Figure S1. Flow cytometric gating strategies for analysis of intra-tumoral T cells in parental and p53KO Colon26 tumors. Supplementary Figure S2. Flow cytometric gating strategies for analysis of intra-tumoral myeloid cells in parental and p53KO Colon26 tumors. Supplementary Figure S3. Anti-tumor activity of HDM201 in Colon26 syngeneic tumor model requires intact immune system. Supplementary Figure S4. p53 knockout in Colon 26 cells results in loss of response to HDM201 in vitro and in vivo. Supplementary Figure S5. HDM201 in combination with PD-1/PD-L1 blockade enhances anti-tumor responses in p53 wild-type tumors, but not in KO tumors. Supplementary Figure S6. Flow cytometric analysis of CD80, GITR, PD-L1, and CD86 expression after HDM201 treatment in vitro. Supplementary Table S1. Antibodies for flow cytometry analysis.

p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell–mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors.Significance:This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.

Half of advanced human melanomas are driven by mutant BRAF and dependent on MAPK signaling. Interestingly, the results of three independent genetic screens highlight a dependency of BRAF-mutant melanoma cell lines on BRAF and ERK2, but not ERK1. ERK2 is expressed higher in melanoma compared with other cancer types and higher than ERK1 within melanoma. However, ERK1 and ERK2 are similarly required in primary human melanocytes transformed with mutant BRAF and are expressed at a similar, lower amount compared with established cancer cell lines. ERK1 can compensate for ERK2 loss as seen by expression of ERK1 rescuing the proliferation arrest mediated by ERK2 loss (both by shRNA or inhibition by an ERK inhibitor). ERK2 knockdown, as opposed to ERK1 knockdown, led to more robust suppression of MAPK signaling as seen by RNA-sequencing, qRT-PCR, and Western blot analysis. In addition, treatment with MAPK pathway inhibitors led to gene expression changes that closely resembled those seen upon knockdown of ERK2 but not ERK1. Together, these data demonstrate that ERK2 drives BRAF-mutant melanoma gene expression and proliferation as a function of its higher expression compared with ERK1. Selective inhibition of ERK2 for the treatment of melanomas may spare the toxicity associated with pan-ERK inhibition in normal tissues. Implications: BRAF-mutant melanomas overexpress and depend on ERK2 but not ERK1, suggesting that ERK2-selective inhibition may be toxicity sparing.

p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell–mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors. Significance: This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.

Uveal melanoma is a rare and aggressive cancer that originates in the eye. Currently, there are no approved targeted therapies and very few effective treatments for this cancer. Although activating mutations in the G protein alpha subunits, GNAQ and GNA11, are key genetic drivers of the disease, few additional drug targets have been identified. Recently, studies have identified context-specific roles for the mammalian SWI/SNF chromatin remodeling complexes (also known as BAF/PBAF) in various cancer lineages. Here, we find evidence that the SWI/SNF complex is essential through analysis of functional genomics screens and further validation in a panel of uveal melanoma cell lines using both genetic tools and small-molecule inhibitors of SWI/SNF. In addition, we describe a functional relationship between the SWI/SNF complex and the melanocyte lineage–specific transcription factor Microphthalmia-associated Transcription Factor, suggesting that these two factors cooperate to drive a transcriptional program essential for uveal melanoma cell survival. These studies highlight a critical role for SWI/SNF in uveal melanoma, and demonstrate a novel path toward the treatment of this cancer.

KRAS, an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells in vitro and in vivo using cell line xenografts and primary human tumors. In vitro, sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in vivo in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and in vivo, suggesting a MAPK pathway–dependent mechanism and possible non-MAPK pathway–dependent mechanisms in tumor cells or tumor microenvironment for the in vivo efficacy. For the KRASG12C MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors.

CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. We compared the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies across multiple cancer cell lines. CRISPR dropout screens consistently identified more lethal genes than RNAi, implying that the identification of many cellular dependencies may require full gene inactivation. However, in two aneuploid cancer models, we found that all genes within highly amplified regions, including nonexpressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits. In addition, using a CRISPR tiling screen, we found that sgRNAs targeting essential domains generate the strongest lethality phenotypes and thus provide a strategy to rapidly define the protein domains required for cancer dependence. Collectively, these findings not only demonstrate the utility of CRISPR screens in the identification of cancer-essential genes, but also reveal the need to carefully control for false-positive results in chromosomally unstable cancer lines. Significance: We show in this study that CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, but have specific liabilities particularly in the interrogation of regions of genome amplification. Therefore, this study provides critical insights for applying CRISPR-based screens toward the systematic identification of new cancer targets. Cancer Discov; 6(8); 900–13. ©2016 AACR. See related commentary by Sheel and Xue, p. 824. See related article by Aguirre et al., p. 914. This article is highlighted in the In This Issue feature, p. 803

The most frequent genetic alterations in melanoma are gain-of-function (GOF) mutations in BRAF, which result in RAF–MEK–ERK signaling pathway addiction. Despite therapeutic success of RAF and MEK inhibitors in treating BRAFV600-mutant tumors, a major challenge is the inevitable emergence of drug resistance, which often involves reactivation of the MAPK pathway. Interestingly, resistant tumors are often sensitive to drug withdrawal, suggesting that hyperactivation of the MAPK pathway is not tolerated. To further characterize this phenomenon, isogenic models of inducible MAPK hyperactivation in BRAFV600E melanoma cells were generated by overexpression of ERK2. Using this model system, supraphysiologic levels of MAPK signaling led to cell death, which was reversed by MAPK inhibition. Furthermore, complete tumor regression was observed in an ERK2-overexpressing xenograft model. To identify mediators of MAPK hyperactivation–induced cell death, a large-scale pooled shRNA screen was conducted, which revealed that only shRNAs against BRAF and MAP2K1 rescued loss of cell viability. This suggested that no single downstream ERK2 effector was required, consistent with pleiotropic effects on multiple cellular stress pathways. Intriguingly, the detrimental effect of MAPK hyperactivation could be partially attributed to secreted factors, and more than 100 differentially secreted proteins were identified. The effect of ERK2 overexpression was highly context dependent, as RAS/RAF mutant but not RAS/RAF wild-type melanoma were sensitive to this perturbation. Implications: This vulnerability to MAPK hyperactivation raises the possibility of novel therapeutic approaches for RAS/RAF-mutant cancers.

Copy number variants (CNVs) were detected and analyzed in 14 probands with autism and intellectual disability with self-injurious behavior (SIB) resulting in tissue damage. For each proband we obtained a clinical history and detailed behavioral descriptions. Genetic anomalies were observed in all probands, and likely clinical significance could be established in four cases. This included two cases having novel, de novo copy number variants and two cases having variants likely to have functional significance. These cases included segmental trisomy 14, segmental monosomy 21, and variants predicted to disrupt the function of ZEB2 (encoding a transcription factor) and HTR2C (encoding a serotonin receptor). Our results identify variants in regions previously implicated in intellectual disability and suggest candidate genes that could contribute to the etiology of SIB.

The most frequent genetic alterations in melanoma are gain-of-function mutations in BRAF, which result in addiction to RAF-MEK-ERK signaling. Given the centrality of this pathway in many cancers, inhibitors have been developed against various nodes in this signaling axis. Despite the success of these inhibitors in treating BRAFV600E mutant tumors, a major problem is the inevitable emergence of drug resistance. The mechanisms of acquired resistance often include reactivation of the MAPK pathway, thus efforts to combat resistance have used vertical combination strategies to inhibit multiple nodes of the pathway. Previous work from our group showed that vemurafenib-resistant melanoma tumors displayed increased levels of MAPK signaling as indicated by elevated phospho-ERK. Strikingly, cell lines derived from these resistant tumors were addicted to vemurafenib, and removal of drug caused loss in cell viability. Consistently, in vivo, the tumors regressed upon cessation of drug treatment, which was accompanied by elevated MAPK signaling. Recent studies have reported similar results for in vitro models of melanoma resistant to MAPK inhibitors, which are addicted to drug in an ERK2-dependent manner. Together these data suggest that extreme levels of MAPK signaling are not tolerated by tumor cells. To further characterize this phenomenon, we generated models of inducible MAPK hyperactivation in BRAFV600E melanoma cells. Using these model systems, we confirmed that increasing the levels of MAPK signaling can hinder tumor cell proliferation, and demonstrated that MAPK inhibitors can rescue this phenotype. Intriguingly, the effect of MAPK hyperactivation varied depending on the RAS/RAF mutation status of the tumor cells. Upon induction of MAPK hyperactivation in sensitive cells, there was no cell cycle arrest, but instead cell death occurred, which was accompanied by activation of caspases. On a global level, MAPK hyperactivation resulted in transcriptome changes that were opposite to those produced by treatment with an ERK inhibitor. This phenomenon of MAPK hyperactivation-induced cell death was also observed in vivo, where complete tumor regression was observed in a xenograft model. Ongoing studies seek to further define the mechanism of cell death, which could suggest potential avenues to leverage hyperactivation of MAPK signaling as a therapeutic approach. Citation Format: Grace P. Leung, Tianshu Feng, Matthew D. Shirley, Frederic D. Sigoillot, David A. Ruddy, Alyson K. Freeman, Mariela Jaskelioff, Darrin D. Stuart. Deleterious effects of MAPK pathway hyperactivation in BRAF-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 965.

The pyfaidx Python module provides memory and time-efficient indexing, subsetting, and in-place modification of subsequences of FASTA files. pyfaidx provides Python classes that expose a dictionary interface where sequences from an indexed FASTA can be accessed by their header name and then sliced by position without reading the full file into memory. pyfaidx includes an extensive test suite to ensure correct and reproducible behavior. A command-line program (faidx) is also provided as an alternative interface, with significant enhancements to functionality, while maintaining full index file compatibility with samtools. The pyfaidx module is installable from PyPI (https://pypi.python.org/pypi/pyfaidx), and development versions can be found at Github (https://github.com/mdshw5/pyfaidx).

The Sturge–Weber syndrome is a sporadic congenital neurocutaneous disorder characterized by a port-wine stain affecting the skin in the distribution of the ophthalmic branch of the trigeminal nerve, abnormal capillary venous vessels in the leptomeninges of the brain and choroid, glaucoma, seizures, stroke, and intellectual disability. It has been hypothesized that somatic mosaic mutations disrupting vascular development cause both the Sturge–Weber syndrome and port-wine stains, and the severity and extent of presentation are determined by the developmental time point at which the mutations occurred. To date, no such mutation has been identified. Methods We performed whole-genome sequencing of DNA from paired samples of visibly affected and normal tissue from 3 persons with the Sturge–Weber syndrome. We tested for the presence of a somatic mosaic mutation in 97 samples from 50 persons with the Sturge–Weber syndrome, a port-wine stain, or neither (controls), using amplicon sequencing and SNaPshot assays, and investigated the effects of the mutation on downstream signaling, using phosphorylation-specific antibodies for relevant effectors and a luciferase reporter assay. Results We identified a nonsynonymous single-nucleotide variant (c.548G→A, p.Arg183Gln) in GNAQ in samples of affected tissue from 88% of the participants (23 of 26) with the Sturge–Weber syndrome and from 92% of the participants (12 of 13) with apparently nonsyndromic port-wine stains, but not in any of the samples of affected tissue from 4 participants with an unrelated cerebrovascular malformation or in any of the samples from the 6 controls. The prevalence of the mutant allele in affected tissues ranged from 1.0 to 18.1%. Extracellular signal-regulated kinase activity was modestly increased during transgenic expression of mutant Gα q. Conclusions The Sturge–Weber syndrome and port-wine stains are caused by a somatic activating mutation in GNAQ. This finding confirms a long-standing hypothesis. (Funded by the National Institutes of Health and Hunter’s Dream for a Cure Foundation.)

A high-resolution map of human phosphorylation networks was constructed by integrating experimentally determined kinase-substrate relationships with other resources, such as in vivo phosphorylation sites., High-quality kinase-substrate relationships (KSRs) were determined using an integrated approach that combines protein microarray technology and bioinformatics analysis. Phosphorylation motifs were predicted for 284 human kinases, representing 55% of the human kinome. A high-resolution map of human phosphorylation networks was constructed that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. A new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling was discovered based on KSRs identified in the phosphorylation networks., The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.

Tens of thousands of lymphoblastoid cell lines (LCLs) have been established by the research community, providing nearly unlimited source material from samples of interest. LCLs are used to address questions in population genomics, mechanisms of disease, and pharmacogenomics. Thus, it is of fundamental importance to define the extent of chromosomal variation in LCLs. We measured variation in genotype and copy number in multiple LCLs derived from peripheral blood mononuclear cells (PBMCs) of single individuals as well as two comparison groups: (1) three types of differentiated cell lines (DCLs) and (2) triplicate HapMap samples. We then validated and extended our findings using data from a large study consisting of samples from blood or LCLs. We observed high concordances between genotypes and copy number estimates within all sample groups. While the genotypes of LCLs tended to faithfully reflect the genotypes of PBMCs, 13.7% (4 of 29) of immortalized cell lines harbored mosaic regions greater than 20 megabases, which were not present in PBMCs, DCLs, or HapMap replicate samples. We created a list of putative LCL-specific changes (affecting regions such as immunoglobulin loci) that is available as a community resource.

Background Mosaic somatic alterations are present in all multi-cellular organisms, but the physiological effects of low-level mosaicism are largely unknown. Most mosaic alterations remain undetectable with current analytical approaches, although the presence of such alterations is increasingly implicated as causative for disease. Results Here, we present the Parent-of-Origin-based Detection (POD) method for chromosomal abnormality detection in trio-based SNP microarray data. Our software implementation, triPOD, was benchmarked using a simulated dataset, outperformed comparable software for sensitivity of abnormality detection, and displayed substantial improvement in the detection of low-level mosaicism while maintaining comparable specificity. Examples of low-level mosaic abnormalities from a large autism dataset demonstrate the benefits of the increased sensitivity provided by triPOD. The triPOD analyses showed robustness across multiple types of Illumina microarray chips. Two large, clinically-relevant datasets were characterized and compared. Conclusions Our method and software provide a significant advancement in the ability to detect low-level mosaic abnormalities, thereby opening new avenues for research into the implications of mosaicism in pathogenic and non-pathogenic processes.