The discovery of RNAi has revolutionized loss-of-function genetic studies in mammalian systems. However, significant challenges still remain to fully exploit RNAi for mammalian genetics. For instance, genetic screens and in vivo studies could be broadly improved by methods that allow inducible and uniform gene expression control. To achieve this, we built the lentiviral pINDUCER series of expression vehicles for inducible RNAi in vivo. Using a multicistronic design, pINDUCER vehicles enable tracking of viral transduction and shRNA or cDNA induction in a broad spectrum of mammalian cell types in vivo. They achieve this uniform temporal, dose-dependent, and reversible control of gene expression across heterogenous cell populations via fluorescence-based quantification of reverse tettransactivator expression. This feature allows isolation of cell populations that exhibit a potent, inducible target knockdown in vitro and in vivo that can be used in human xenotransplantation models to examine cancer drug targets.lentivirus | vector L oss-of-function studies represent a powerful means by which to gain insight into the molecular mechanisms behind complex biological processes. Until recently, these studies were practical for only a small set of genetically tractable model organisms (1, 2). The discovery of gene silencing through RNAi has made it possible to carry out loss-of-function studies in mammals. RNAi has transformed the way in which gene function can be investigated and has quickly become a versatile tool for a wide range of applications, including reverse genetics, highthroughput screens, and therapeutics (3). The application of RNAi technology both in vitro and in vivo has tremendous potential to further our knowledge of the molecular mechanisms that underpin both normal biology and human disease.To achieve efficient and long-term gene silencing, we have previously generated the retroviral-encoded microRNA-based shRNA PRIME (potent RNA interference using microRNA expression) vectors that are effective in suppressing expression of their intended gene targets from single proviral integrations (4). These vectors use the miR30 backbone and are expressed from RNA polymerase II promoters, which allows for exquisite regulation of shRNA expression. We have further generated genomewide shRNA libraries in these vectors that cover large numbers of mammalian transcripts for systematic study of gene function (5, 6). These shRNA libraries, as well as shRNA libraries generated by other laboratories (7,8), have been used to perform genetic screens in mammalian cell culture models for a variety of phenotypes, including cell transformation, synthetic lethal interactions, and resistance to chemotherapeutic treatments (9-15).Despite the success demonstrated by the above constitutive shRNA vectors, an inducible shRNA system would have obvious advantages in many experimental settings. First, the inducible shRNA system allows for the study of essential genes. Because constitutively expressed shRNAs targeting essential genes will be ...

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.

SUMMARY Among breast cancers, triple-negative breast cancer (TNBC) is the most poorly understood and is refractory to current targeted therapies. Using a genetic screen, we identify the PTPN12 tyrosine phosphatase as a tumor suppressor in TNBC. PTPN12 potently suppresses mammary epithelial cell proliferation and transformation. PTPN12 is frequently compromised in human TNBCs, and we identify an upstream tumor-suppressor network that posttranscriptionally controls PTPN12. PTPN12 suppresses transformation by interacting with and inhibiting multiple oncogenic tyrosine kinases, including HER2 and EGFR. The tumorigenic and metastatic potential of PTPN12-deficient TNBC cells is severely impaired upon restoration of PTPN12 function or combined inhibition of PTPN12-regulated tyrosine kinases, suggesting that TNBCs are dependent on the proto-oncogenic tyrosine kinases constrained by PTPN12. Collectively, these data identify PTPN12 as a commonly inactivated tumor suppressor and provide a rationale for combinatorially targeting proto-oncogenic tyrosine kinases in TNBC and other cancers based on their profile of tyrosine-phosphatase activity.

During interphase in all eukaryotic cells the double lipid bilayer of the nuclear envelope (NE) physically separates the chromosomes, and chromosome-related processes, from the cytoplasm and increases in area by 59% (Lim et al., 2007) as the nuclear volume doubles in preparation for mitosis (reviewed by Hetzer et al., 2005;Lim et al., 2007;Winey et al., 1997). In the open mitosis of animal cells, NE breakdown allows the spindle microtubules that are nucleated by the cytoplasmic centrosomes to attach to and then separate the chromosomes. In the closed mitosis of yeast, the centrosome equivalents, called spindle pole bodies (SPBs), are embedded in the NE and nucleate the formation of an intranuclear spindle (Ding et al., 1997). As the spindle elongates in anaphase B, nuclear volume remains constant but division of the roughly spherical nucleus into two smaller spheres, which occurs in less than 5 minutes, requires a rapid increase of 26% in NE area (Lim et al., 2007).The nucleus, often thought of as a freestanding organelle, is actually a specialized region of the endoplasmic reticulum (ER) (Voeltz et al., 2002): the outer NE is continuous with both the ER and the inner NE (Hetzer et al., 2005), providing a means by which lipids and membrane proteins can move between the sheet form of the ER at the nuclear periphery and the tubular form of the ER, which, in animal cells, extends throughout the cytoplasm and, in yeast, is primarily at the cell periphery (Pidoux and Armstrong, 1993;Voeltz et al., 2002).The Ran GTPase influences many cellular functions (Quimby and Dasso, 2003), including nucleocytoplasmic transport, NE reformation after mitosis in animal cells (Hetzer et al., 2005), and NE structure in fission (Demeter et al., 1995) and budding (Ryan et al., 2003) yeast. However, the mechanism by which the Ran GTPase influences mitosis-specific NE changes during both open and closed mitosis remains unknown. We have previously shown that, when the Ran system is mis-regulated in the fission yeast Schizosaccharomyces pombe, cells arrest with NEs that have lost their integrity, and that this defect is concomitant with and depends on the passage through mitosis (Demeter et al., 1995).The predictions of a biophysical model of the fission-yeast nucleus (Lim et al., 2007) and experimental observations of abnormal nuclear shapes seen when microtubule bundles lacking SPBs at their ends cause thin tethers to protrude from the spherical nucleus (Khodjakov et al., 2004;Tange et al., 2002;Toya et al., 2007;West et al., 1998;Zheng et al., 2007) raise the possibility that the SPB influences the mechanical properties of the NE to prevent tether formation during normal mitosis and ensure symmetric nuclear division. Because of limitations on the mechanical strength of lipid bilayers and their ability to stretch in response to pressure exerted by the elongating spindle, our computational model incorporates a lipid reservoir (Lim et al.,The double lipid bilayer of the nuclear envelope (NE) remains intact during closed mitosis. In the ...

Myc is an oncogenic transcription factor frequently dysregulated in human malignancies. While the transcriptional programs and other functions of Myc have been intensively studied, there remains no effective strategy for inhibiting Myc in patients. To search for pathways that support the Myc oncogenic program, we employed a next-generation RNAi screen for Myc-synthetic lethal (MySL) genes. Using this strategy, we have identified several cellular processes required to tolerate oncogenic Myc. Key among these is the core sumoylation machinery, and we define the Sumo-activating enzyme (SAE) as a central component in this MySL network. Loss of SAE drives synthetic lethality with Myc, and the enzymatic activity of SAE is required to support the Myc oncogenic state. Inactivation of SAE leads to mitotic catastrophe and cell death selectively upon Myc hyper-activation. Mechanistically, depletion of SAE switches a subprogram of Myc transcriptional targets governing mitotic spindle function from activated to repressed, a subprogram we term Sumoylation-dependent Myc Switchers, or SMS genes. Notably, SMS genes are required to tolerate Myc hyper-activation, and SAE and the SMS program are required for Myc-dependent breast cancer cell survival in vitro and tumor growth and progression in vivo. Importantly, patient survival significantly correlates with levels of SAE and SMS gene expression in Myc-high tumors. Collectively, these studies reveal a mitotic vulnerability of Myc-driven cancers and demonstrate that inhibiting sumoylation can selectively impair mitosis and survival in an oncogenic Myc-dependent manner. We propose that drugs targeting SAE and its downstream SMS targets may have therapeutic benefits for patients with Myc-driven cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3091. doi:1538-7445.AM2012-3091