Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we employed a genome-wide RNAi screen for Myc-synthetic-lethal genes and uncovered a role for the SUMO-activating-enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death selectively upon Myc hyper-activation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent-Myc-switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for Myc-dependent tumor growth, and patient survival significantly correlates with SAE1/SAE2 levels in Myc-high tumors. These studies reveal a mitotic vulnerability of Myc-driven cancers, demonstrate that inhibiting sumoylation impairs Myc-dependent tumorigenesis, and suggest inhibiting SUMOylation may have therapeutic benefits for patients with Myc-driven cancer.

Tumorigenesis is a multistep process characterized by a myriad of genetic and epigenetic alterations. Identifying the causal perturbations that confer malignant transformation is a central goal in cancer biology. Here we report an RNAi-based genetic screen for genes that suppress transformation of human mammary epithelial cells. We identified genes previously implicated in proliferative control and epithelial cell function including two established tumor suppressors, TGFBR2 and PTEN. In addition, we uncovered a previously unrecognized tumor suppressor role for REST/NRSF, a transcriptional repressor of neuronal gene expression. Array-CGH analysis identified REST as a frequent target of deletion in colorectal cancer. Furthermore, we detect a frameshift mutation of the REST gene in colorectal cancer cells that encodes a dominantly acting truncation capable of transforming epithelial cells. Cells lacking REST exhibit increased PI(3)K signaling and are dependent upon this pathway for their transformed phenotype. These results implicate REST as a human tumor suppressor and provide a novel approach to identifying candidate genes that suppress the development of human cancer.

Retroviral short hairpin RNA (shRNA)-mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.We have recently generated barcoded, microRNA-based shRNA libraries targeting the entire human genome that can be expressed efficiently from retroviral or lenti-viral vectors in a variety of cell types for stable gene knockdown (1,2). Furthermore, we have also developed a method of screening complex pools of shRNAs using barcodes coupled with microarray deconvolution to take advantage of the highly parallel format, low cost, and flexibility in assay design of this approach (2,3). Although barcodes are not essential for enrichment screens (positive selection) (3-5), they are critical for dropout screens (negative selection) such as those designed to identify cell-lethal or drug-sensitive shRNAs (6). Hairpins that are depleted over time can be † To whom correspondence should be addressed. selledge@genetics.med.harvard.edu. * These authors contributed equally to this work. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript identified through the competitive hybridization of barcodes derived from the shRNA population before and after selection to a microarray (Fig. 1A).We previously described the use of 60-nucleotide barcodes for pool deconvolution (2,3). To provide an alternative to these barcodes that enables a more rapid construction and screening of shRNA libraries, we have developed a methodology called half-hairpin (HH) barcoding for deconvoluting pooled shRNAs (7). We took advantage of the large 19-nucleotide hairpin loop of our mir30-based platform and designed a polymerase chain reaction (PCR) strategy that amplifies only the 3′ half of the shRNA stem (Fig. 1B). As compared with full-hairpin sequences for microarray hybridization (8,9), HH barcodes entirely eliminate probe selfannealing during microarray hybridization (Fig. 1C and fig. S1, A and B), providing the critical dynamic range necessary for pool-based dropout screens. HH barcode signals are highly reproducible in replicate PCRs (R = 0.973, fig. S1A), highly specific (0.5% cross-reaction) ( fig. S1C), and display a reasonable, although slightly compressed, dynamic range in mixing experiments w...

The transcription factor REST/NSRF (RE1-Silencing Transcription Factor) is a master repressor of neuronal gene expression and neuronal programs in non-neuronal lineages 1 − 3 . Recently, REST was identified as a human tumor suppressor in epithelial tissues 4 , suggesting that REST regulation may have important physiologic and pathologic consequences. However, the pathways controlling REST have yet to be elucidated. Here, we demonstrate that REST is regulated by ubiquitin-mediated proteolysis, and use an RNAi screen to identify SCF βTRCP as an E3 ubiquitin ligase responsible for REST degradation. βTRCP binds and ubiquitinates REST and controls its stability through a conserved phosphodegron. During neural differentiation REST is degraded in a βTRCP-dependent manner. βTRCP is required for proper neural differentiation only in the presence of REST, indicating that βTRCP facilitates this process through degradation of REST. Conversely, failure to degrade REST attenuates differentiation. Furthermore, we find that βTRCP overexpression, which is common in human epithelial cancers, causes oncogenic transformation of human mammary epithelial cells and this pathogenic function requires REST degradation. Thus, REST is a key target in βTRCP-driven transformation and the βTRCP-REST axis is a new regulatory pathway controlling neurogenesis.REST levels decline during differentiation of embryonic stem cells to neural stem and progenitor cells 5 , consistent with a role for REST in restraining neuronal gene expression programs. This decrease results from a 3-fold reduction in REST half-life (Fig. 1a), suggesting that a regulatory pathway controls REST degradation during early neural differentiation. To determine whether ubiquitination is involved, REST was evaluated for ubiquitin-modification in vivo. Immunoprecipitation of HA-ubiquitin revealed slower migrating species of REST suggestive of polyubiquitination (Fig. 1b, lane 3 (Fig. 1b, lane 4), suggesting REST is K48 polyubiquitinated which promotes degradation.To search for the E3 ubiquitin ligase for REST, we began with the SCF superfamily of ligases 6 . Each SCF family contains a common Cullin scaffold that is required for ligase function. Notably, coexpression of a dominant negative Cullin-1 (Cul1) mutant resulted in a dramatic increase (11-fold) in REST levels (Supp. Fig. 1b), indicating that one or more Cul1-containing ligases negatively regulate REST abundance.F-box proteins act as substrate receptors for the SCF 7,8 . To determine which F-box proteins are required for REST turnover, we established a system for monitoring REST abundance in a high-throughput manner using an mRFP-REST fusion protein. Similar to endogenous REST, mRFP-REST was unstable, and its abundance increased upon inhibition of Cul-1 (Supp. Fig. 2a). To identify the F-box proteins regulating REST, individual siRNAs targeting each F-box protein (4 siRNAs/gene) were cotransfected with a plasmid encoding mRFP-REST, and changes in cellular fluorescence were monitored by flow cytometry (Supp. Fig. 2...

Tumors exhibit numerous recurrent hemizygous focal deletions that contain no known tumor suppressors and are poorly understood. To investigate whether these regions contribute to tumorigenesis, we searched genetically for genes with cancer-relevant properties within these hemizygous deletions. We identified STOP and GO genes, which negatively and positively regulate proliferation, respectively. STOP genes include many known tumor suppressors, whereas GO genes are enriched for essential genes. Analysis of their chromosomal distribution revealed that recurring deletions preferentially over represent STOP genes and under represent GO genes. We propose a hypothesis called the cancer gene island model whereby gene islands encompassing high densities of STOP genes and low densities of GO genes are hemizygously deleted to maximize proliferative fitness through cumulative haploinsufficiencies. Because hundreds to thousands of genes are hemizygously deleted per tumor, this mechanism may help drive tumorigenesis across many cancer types.

Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify new DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors and >70% of randomly tested transcription factors localized to sites of DNA damage and approximately 90% were PARP-dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding domain-dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP-dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.

Oligodendrocyte precursor cells (OPCs) in the adult brain contribute to white matter homeostasis. After white matter damage, OPCs compensate for oligodendrocyte loss by differentiating into mature oligodendrocytes. However, the underlying mechanisms remain to be fully defined. Here, we test the hypothesis that, during endogenous recovery from white matter ischemic injury, astrocytes support the maturation of OPCs by secreting brain-derived neurotrophic factor (BDNF). For in vitro experiments, cultured primary OPCs and astrocytes were prepared from postnatal day 2 rat cortex. When OPCs were subjected to chemical hypoxic stress by exposing them to sublethal CoCl 2 for 7 d, in vitro OPC differentiation into oligodendrocytes was significantly suppressed. Conditioned medium from astrocytes (astro-medium) restored the process of OPC maturation even under the stressed conditions. When astro-medium was filtered with TrkB-Fc to remove BDNF, the BDNF-deficient astro-medium no longer supported OPC maturation. For in vivo experiments, we analyzed a transgenic mouse line (GFAP cre /BDNF wt/fl ) in which BDNF expression is downregulated specifically in GFAP ϩ astrocytes. Both wild-type (GFAP wt /BDNF wt/fl mice) and transgenic mice were subjected to prolonged cerebral hypoperfusion by bilateral common carotid artery stenosis. As expected, compared with wild-type mice, the transgenic mice exhibited a lower number of newly generated oligodendrocytes and larger white matter damage. Together, these findings demonstrate that, during endogenous recovery from white matter damage, astrocytes may promote oligodendrogenesis by secreting BDNF.