The adaptive immune system is a major driver of selection for tumor suppressor gene inactivation.

During tumorigenesis, tumors must evolve to evade the immune system and do so by disrupting the genes involved in antigen processing and presentation or up-regulating inhibitory immune checkpoint genes. We performed in vivo CRISPR screens in syngeneic mouse tumor models to examine requirements for tumorigenesis both with and without adaptive immune selective pressure. In each tumor type tested, we found a marked enrichment for the loss of tumor suppressor genes (TSGs) in the presence of an adaptive immune system relative to immunocompromised mice. Nearly one-third of TSGs showed preferential enrichment, often in a cancer- and tissue-specific manner. These results suggest that clonal selection of recurrent mutations found in cancer is driven largely by the tumor's requirement to avoid the adaptive immune system.

CARM1 Inhibition Enables Immunotherapy of Resistant Tumors by Dual Action on Tumor Cells and T Cells.

A number of cancer drugs activate innate immune pathways in tumor cells but unfortunately also compromise antitumor immune function. We discovered that inhibition of CARM1, an epigenetic enzyme and cotranscriptional activator, elicited beneficial antitumor activity in both cytotoxic T cells and tumor cells. In T cells, Carm1 inactivation substantially enhanced their antitumor function and preserved memory-like populations required for sustained antitumor immunity. In tumor cells, Carm1 inactivation induced a potent type 1 interferon response that sensitized resistant tumors to cytotoxic T cells. Substantially increased numbers of dendritic cells, CD8 T cells, and natural killer cells were present in Carm1-deficient tumors, and infiltrating CD8 T cells expressed low levels of exhaustion markers. Targeting of CARM1 with a small molecule elicited potent antitumor immunity and sensitized resistant tumors to checkpoint blockade. Targeting of this cotranscriptional regulator thus offers an opportunity to enhance immune function while simultaneously sensitizing resistant tumor cells to immune attack. SIGNIFICANCE: Resistance to cancer immunotherapy remains a major challenge. Targeting of CARM1 enables immunotherapy of resistant tumors by enhancing T-cell functionality and preserving memory-like T-cell populations within tumors. CARM1 inhibition also sensitizes resistant tumor cells to immune attack by inducing a tumor cell-intrinsic type 1 interferon response.This article is highlighted in the In This Issue feature, p. 1861.

Diversified Application of Barcoded PLATO (PLATO-BC) Platform for Identification of Protein Interactions.

Proteins usually associate with other molecules physically to execute their functions. Identifying these interactions is important for the functional analysis of proteins. Previously, we reported the parallel analysis of translated ORFs (PLATO) to couple ribosome display of full-length ORFs with affinity enrichment of mRNA/protein/ribosome complexes for the "bait" molecules, followed by the deep sequencing analysis of mRNA. However, the sample processing, from extraction of precipitated mRNA to generation of DNA libraries, includes numerous steps, which is tedious and may cause the loss of materials. Barcoded PLATO (PLATO-BC), an improved platform was further developed to test its application for protein interaction discovery. In this report, we tested the antisera-antigen interaction using serum samples from patients with inclusion body myositis (IBM). Tripartite motif containing 21 (TRIM21) was identified as a potentially new IBM autoantigen. We also expanded the application of PLATO-BC to identify protein interactions for JQ1, single ubiquitin peptide, and NS5 protein of Zika virus. From PLATO-BC analyses, we identified new protein interactions for these "bait" molecules. We demonstrate that Ewing sarcoma breakpoint region 1 (EWSR1) binds to JQ1 and their interactions may interrupt the EWSR1 association with acetylated histone H4. RIO kinase 3 (RIOK3), a newly identified ubiquitin-binding protein, is preferentially associated with K63-ubiquitin chain. We also find that Zika NS5 protein interacts with two previously unreported host proteins, par-3 family cell polarity regulator (PARD3) and chromosome 19 open reading frame 53 (C19orf53), whose attenuated expression benefits the replication of Zika virus. These results further demonstrate that PLATO-BC is capable of identifying novel protein interactions for various types of "bait" molecules.

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

Profound Tissue Specificity in Proliferation Control Underlies Cancer Drivers and Aneuploidy Patterns.

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.

A Role for Mitochondrial Translation in Promotion of Viability in K-Ras Mutant Cells.

Activating mutations in the KRAS oncogene are highly prevalent in tumors, especially those of the colon, lung, and pancreas. To better understand the genetic dependencies that K-Ras mutant cells rely upon for their growth, we employed whole-genome CRISPR loss-of-function screens in two isogenic pairs of cell lines. Since loss of essential genes is uniformly toxic in CRISPR-based screens, we also developed a small hairpin RNA (shRNA) library targeting essential genes. These approaches uncovered a large set of proteins whose loss results in the selective reduction of K-Ras mutant cell growth. Pathway analysis revealed that many of these genes function in the mitochondria. For validation, we generated isogenic pairs of cell lines using CRISPR-based genome engineering, which confirmed the dependency of K-Ras mutant cells on these mitochondrial pathways. Finally, we found that mitochondrial inhibitors reduce the growth of K-Ras mutant tumors in vivo, aiding in the advancement of strategies to target K-Ras-driven malignancy.

A genetic interaction analysis identifies cancer drivers that modify EGFR dependency.

A large number of cancer drivers have been identified through tumor sequencing efforts, but how they interact and the degree to which they can substitute for each other have not been systematically explored. To comprehensively investigate how cancer drivers genetically interact, we searched for modifiers of epidermal growth factor receptor (EGFR) dependency by performing CRISPR, shRNA, and expression screens in a non-small cell lung cancer (NSCLC) model. We elucidated a broad spectrum of tumor suppressor genes (TSGs) and oncogenes (OGs) that can genetically modify proliferation and survival of cancer cells when EGFR signaling is altered. These include genes already known to mediate EGFR inhibitor resistance as well as many TSGs not previously connected to EGFR and whose biological functions in tumorigenesis are not well understood. We show that mutation of PBRM1, a subunit of the SWI/SNF complex, attenuates the effects of EGFR inhibition in part by sustaining AKT signaling. We also show that mutation of Capicua (CIC), a transcriptional repressor, suppresses the effects of EGFR inhibition by partially restoring the EGFR-promoted gene expression program, including the sustained expression of Ets transcription factors such as ETV1 Together, our data provide strong support for the hypothesis that many cancer drivers can substitute for each other in certain contexts and broaden our understanding of EGFR regulation.

Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer.

Scleroderma is a chronic autoimmune rheumatic disease associated with widespread tissue fibrosis and vasculopathy. Approximately two-thirds of all patients with scleroderma present with three dominant autoantibody subsets. Here, we used a pair of complementary high-throughput methods for antibody epitope discovery to examine patients with scleroderma with or without known autoantibody specificities. We identified a specificity for the minor spliceosome complex containing RNA Binding Region (RNP1, RNA recognition motif) Containing 3 (RNPC3) that is found in patients with scleroderma without known specificities and is absent in unrelated autoimmune diseases. We found strong evidence for both intra- and intermolecular epitope spreading in patients with RNA polymerase III (POLR3) and the minor spliceosome specificities. Our results demonstrate the utility of these technologies in rapidly identifying antibodies that can serve as biomarkers of disease subsets in the evolving precision medicine era.

Functional genomics reveals that tumors with activating phosphoinositide 3-kinase mutations are dependent on accelerated protein turnover.

Activating mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway are frequently identified in cancer. To identify pathways that support PI3K oncogenesis, we performed a genome-wide RNAi screen in isogenic cell lines harboring wild-type or mutant PIK3CA to search for PI3K synthetic-lethal (SL) genes. A combined analysis of these results with a meta-analysis of two other large-scale RNAi screening data sets in PI3K mutant cancer cell lines converged on ribosomal protein translation and proteasomal protein degradation as critical nononcogene dependencies for PI3K-driven tumors. Genetic or pharmacologic inhibition of either pathway alone, but not together, selectively killed PI3K mutant tumor cells in an mTOR-dependent manner. The expression of ribosomal and proteasomal components was significantly up-regulated in primary human colorectal tumors harboring PI3K pathway activation. Importantly, a PI3K SL gene signature containing the top hits of the SL genes identified in our meta-analysis robustly predicted overall patient survival in colorectal cancer, especially among patients with tumors with an activated PI3K pathway. These results suggest that disruption of protein turnover homeostasis via ribosome or proteasome inhibition may be a novel treatment strategy for PI3K mutant human tumors.

The DNA damage response induces inflammation and senescence by inhibiting autophagy of GATA4.

Cellular senescence is a terminal stress-activated program controlled by the p53 and p16(INK4a) tumor suppressor proteins. A striking feature of senescence is the senescence-associated secretory phenotype (SASP), a pro-inflammatory response linked to tumor promotion and aging. We have identified the transcription factor GATA4 as a senescence and SASP regulator. GATA4 is stabilized in cells undergoing senescence and is required for the SASP. Normally, GATA4 is degraded by p62-mediated selective autophagy, but this regulation is suppressed during senescence, thereby stabilizing GATA4. GATA4 in turn activates the transcription factor NF-κB to initiate the SASP and facilitate senescence. GATA4 activation depends on the DNA damage response regulators ATM and ATR, but not on p53 or p16(INK4a). GATA4 accumulates in multiple tissues, including the aging brain, and could contribute to aging and its associated inflammation.

STOP gene Phactr4 is a tumor suppressor.

Cancer develops through genetic and epigenetic alterations that allow unrestrained proliferation and increased survival. Using a genetic RNAi screen, we previously identified hundreds of suppressors of tumorigenesis and/or proliferation (STOP) genes that restrain normal cell proliferation. Our STOP gene set was significantly enriched for known and putative tumor suppressor genes. Here, we report a tumor-suppressive role for one STOP gene, phosphatase and actin regulator 4 (PHACTR4). Phactr4 is one of four members of the largely uncharacterized Phactr family of protein phosphatase 1 (PP1)-and actin-binding proteins. Our work suggests that Phactr4 restrains normal cell proliferation and transformation. Depletion of Phactr4 with multiple shRNAs leads to increased proliferation and soft agar colony formation. Phactr4 acts, in part, through an Rb-dependent pathway, because Rb phosphorylation is maintained upon growth factor withdrawal in Phactr4-depleted cells. Examination of tumor copy number analysis and sequencing revealed that PHACTR4 is significantly deleted and mutant in many tumor subtypes. Furthermore,cancer cell lines with reduced Phactr4 expression exhibit tumor suppressor hypersensitivity upon Phactr4 complementation,leading to reduced proliferation, transformation, and tumor formation. Thus, Phactr4 acts as a tumor suppressor that is deleted and mutant in several cancers.

Recurrent hemizygous deletions in cancers may optimize proliferative potential.

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 overrepresent STOP genes and underrepresent 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 to drive tumorigenesis across many cancer types.

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy.

shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs.

A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis.

Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search 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 upon Myc hyperactivation. 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 growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.

Autoantigen discovery with a synthetic human peptidome.

Immune responses targeting self-proteins (autoantigens) can lead to a variety of autoimmune diseases. Identification of these antigens is important for both diagnostic and therapeutic reasons. However, current approaches to characterize autoantigens have, in most cases, met only with limited success. Here we present a synthetic representation of the complete human proteome, the T7 peptidome phage display library (T7-Pep), and demonstrate its application to autoantigen discovery. T7-Pep is composed of >413,000 36-residue, overlapping peptides that cover all open reading frames in the human genome, and can be analyzed using high-throughput DNA sequencing. We developed a phage immunoprecipitation sequencing (PhIP-Seq) methodology to identify known and previously unreported autoantibodies contained in the spinal fluid of three individuals with paraneoplastic neurological syndromes. We also show how T7-Pep can be used more generally to identify peptide-protein interactions, suggesting the broader utility of our approach for proteomic research.

Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase.

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.

The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo.

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 tet-transactivator 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.

Cancer proliferation gene discovery through functional genomics.

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.

Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities.

The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2-Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2-Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.