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PDF file 1227K, This file contains Supplemental Figures 1-7, which provide additional supporting information for each of the main figures

Copy number variation (CNV) of samples in TCGA

Supplementary Table S7 - XLSX file 61K, List of shRNAs used in this study

DEDD and MCL1 overexpression are not necessary for initiation of tumorigenesis.

Supplementary Figure 2 - PDF file 17K, SKIL induces Matrigel invasion

PDF file 146K, This file contains the legends to Supplementary Figures 1-7 and Supplementary methods

S1 XLSX file 63K, This Supplementary Table contains the results of ORF screen to identify genes that can rescue the suppression of TBK1 in a KRAS-dependent cell line

Focal amplification of chromosome 1q23.3 in patients with advanced primary or relapsed urothelial carcinomas is associated with poor survival. We interrogated chromosome 1q23.3 and the nearby focal amplicon 1q21.3, as both are associated with increased lymph node disease in patients with urothelial carcinoma. Specifically, we assessed whether the oncogene MCL1 that resides in 1q21.3 and the genes that reside in the 1q23.3 amplicon were required for the proliferation or survival of urothelial carcinoma. We observed that suppressing MCL1 or the death effector domain–containing protein (DEDD) in the cells that harbor amplifications of 1q21.3 or 1q23.3, respectively, inhibited cell proliferation. We also found that overexpression of MCL1 or DEDD increased anchorage independence growth in vitro and increased experimental metastasis in vivo in the nonamplified urothelial carcinoma cell line, RT112. The expression of MCL1 confers resistance to a range of apoptosis inducers, while the expression of DEDD led to resistance to TNFα-induced apoptosis. These observations identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinoma.Implications:These studies identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinomas.

Supplementary Legends - PDF file 122K, Legends for all Supplementary information

Supplementary Figure 3 - PDF file 20K, 3q26 copy number in cancer cell lines

Supplementary Figure 1 - PDF file 25K, Correlation between TLOC1 suppression and cell proliferation

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753–65. ©2016 AACR.

Description of additional methods and procedures used in the study.

GO terms for NEK6 upregulated genes Day 5 after castration (filtered for >10 GeneHits).

LISA analysis of master regulators of differentially expressed genes

Supplementary Data including supplementary methods, supplementary figures, and supplementary figure legends.

GSEA Hallmarks analysis of RNA-seq of bulk lung tumors

RNA-seq data of bulk lung tumors from sgControl RPP, sgNotch1 RPP, sgNotch2 RPP, and sgAscl1 RPP CRISPR-based SCLC GEMMs

RNA-seq data of the SCLC adherent cells that are either Notch-WT or Notch-Mutant

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations.Significance:A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST–dependent pathway and innate immune signaling.

Legend for Supplemental Figures S1-S6.

Neuroendocrine to nonneuroendocrine plasticity supports small cell lung cancer (SCLC) tumorigenesis and promotes immunogenicity. Approximately 20% to 25% of SCLCs harbor loss-of-function (LOF) NOTCH mutations. Previous studies demonstrated that NOTCH functions as a SCLC tumor suppressor, but can also drive nonneuroendocrine plasticity to support SCLC growth. Given the dual functionality of NOTCH, it is not understood why SCLCs select for LOF NOTCH mutations and how these mutations affect SCLC tumorigenesis. In a CRISPR-based genetically engineered mouse model of SCLC, genetic loss of Notch1 or Notch2 modestly accelerated SCLC tumorigenesis. Interestingly, Notch-mutant SCLCs still formed nonneuroendocrine subpopulations, and these Notch-independent, nonneuroendocrine subpopulations were driven by Runx2-mediated regulation of Rest. Notch2-mutant nonneuroendocrine cells highly express innate immune signaling genes including stimulator of interferon genes (STING) and were sensitive to STING agonists. This work identifies a Notch-independent mechanism to promote nonneuroendocrine plasticity and suggests that therapeutic approaches to activate STING could be selectively beneficial for SCLCs with NOTCH2 mutations. Significance: A genetically engineered mouse model of NOTCH-mutant SCLC reveals that nonneuroendocrine plasticity persists in the absence of NOTCH, driven by a RUNX2-REST–dependent pathway and innate immune signaling.

Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that mediates integrin and Fc receptor signaling in myeloid cells. SYK has been implicated as an oncogenic driver in acute myeloid leukemia (AML) with aberrant expression of HOXA9 and MEIS1 and cooperates with FLT3 internal tandem duplication to drive leukemogenesis. The oral SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity in HOXA9/MEIS1 driven AML and is currently being investigated in a phase 3 trial of previously untreated patients with nucleophosmin1-mutated (NPM1 mut) AML. Lanraplenib (LANRA) is a next generation oral SYK inhibitor with potency and selectivity comparable to ENTO. In healthy volunteers and patients with autoimmune disease, LANRA has shown pharmacokinetic properties that compare favorably with ENTO. To support the clinical development of LANRA for the treatment of AML, ex vivo treatment of patient-derived AML cells was used to compare its activity to that of ENTO, both as a single-agent and in combination with other AML therapies. First, ENTO and LANRA single-agent activities were evaluated in peripheral blood-derived blasts from 15 AML patients, representing different mutational backgrounds including NPM1, FLT3, PTPN11, and NRAS mutations. AML cells were seeded into 96 well plates and treated with ENTO and LANRA for 6 days. Comparable effects on viability were observed across the 15 models with the 2 compounds, and in 11 of the models, the half maximal inhibitory concentration (IC 50) values were within 2-fold of each other. ENTO had a slightly lower IC 50 value than LANRA in the FLT3-mutated models possibly due to the direct FLT3 inhibitory activity of ENTO. Next, we tested the activity of ENTO and LANRA ex vivo in bone marrow-derived AML blasts from 29 AML patients representing diverse mutational backgrounds, including NPM1, IDH1, FLT3, and RAS mutations as well as MLL rearrangements. The models were treated for 9 days with either ENTO or LANRA, and viability was assessed using Annexin V and 7-aminoactinomycin D staining. Again, ENTO and LANRA showed comparable effects on cell viability with no significant differences between the compounds when compared across the different mutational backgrounds. Both studies suggest the potential for anti-leukemic activity in several different genetically defined subsets of AML. Matrix combination assays were performed by combining ENTO or LANRA with either cytarabine (NPM1 mut), gilteritinib (FLT3 mut), or trametinib (RAS mut) with cell viability and death assessed after a 3-day incubation period. Increased cell death in an additive manner was observed in all combinations tested, with results for ENTO and LANRA being similar, indicating the utility of both compounds in combinatorial treatment paradigms. These results support the clinical evaluation of LANRA in genetically defined subsets of AML. A phase 1b/2 study of LANRA in combination with the selective FLT3 inhibitor gilteritinib, in patients with relapsed or refractory FLT3 mut AML is planned for the end of this year. Disclosures Day: Cyteir Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Heckman: Novartis: Research Funding; Orion Pharma: Research Funding; Celgene/BMS: Research Funding; Oncopeptides: Consultancy, Research Funding; Kronos Bio, Inc.: Research Funding. Schinzel: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Obholzer: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Lin: Kronos Bio, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Saffran: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company.

Chromosomal rearrangements are essential events in the pathogenesis of both malignant and nonmalignant disorders, yet the factors affecting their formation are incompletely understood. Here we develop a zinc-finger nuclease translocation reporter and screen for factors that modulate rearrangements in human cells. We identify UBC9 and RAD50 as suppressors and 53BP1, DDB1 and poly(ADP)ribose polymerase 3 (PARP3) as promoters of chromosomal rearrangements across human cell types. We focus on PARP3 as it is dispensable for murine viability and has druggable catalytic activity. We find that PARP3 regulates G quadruplex (G4) DNA in response to DNA damage, which suppresses repair by nonhomologous end-joining and homologous recombination. Chemical stabilization of G4 DNA in PARP3−/− cells leads to widespread DNA double-strand breaks and synthetic lethality. We propose a model in which PARP3 suppresses G4 DNA and facilitates DNA repair by multiple pathways., Chromosomal rearrangements are key events in the pathogenesis of a range of disorders. Here the authors utilize a zinc finger nuclease translocation reporter to identify PARP3 as a regulator of these events at sites enriched for G quadruplex DNA.

More than 90% of small cell lung cancers (SCLCs) harbor loss-of-function mutations in the tumor suppressor gene RB1. The canonical function of the RB1 gene product, pRB, is to repress the E2F transcription factor family, but pRB also functions to regulate cellular differentiation in part through its binding to the histone demethylase KDM5A (also known as RBP2 or JARID1A). We show that KDM5A promotes SCLC proliferation and SCLC's neuroendocrine differentiation phenotype in part by sustaining expression of the neuroendocrine transcription factor ASCL1. Mechanistically, we found that KDM5A sustains ASCL1 levels and neuroendocrine differentiation by repressing NOTCH2 and NOTCH target genes. To test the role of KDM5A in SCLC tumorigenesis in vivo, we developed a CRISPR/Cas9-based mouse model of SCLC by delivering an adenovirus (or an adeno-associated virus [AAV]) that expresses Cre recombinase and sgRNAs targeting Rb1, Tp53, and Rbl2 into the lungs of Lox-Stop-Lox Cas9 mice. Coinclusion of a KDM5A sgRNA decreased SCLC tumorigenesis and metastasis, and the SCLCs that formed despite the absence of KDM5A had higher NOTCH activity compared to KDM5A+/+ SCLCs. This work establishes a role for KDM5A in SCLC tumorigenesis and suggests that KDM5 inhibitors should be explored as treatments for SCLC.

Focal amplification of chromosome 1q23.3 in patients with advanced primary or relapsed urothelial carcinomas is associated with poor survival. We interrogated chromosome 1q23.3 and the nearby focal amplicon 1q21.3, as both are associated with increased lymph node disease in patients with urothelial carcinoma. Specifically, we assessed whether the oncogene MCL1 that resides in 1q21.3 and the genes that reside in the 1q23.3 amplicon were required for the proliferation or survival of urothelial carcinoma. We observed that suppressing MCL1 or the death effector domain–containing protein (DEDD) in the cells that harbor amplifications of 1q21.3 or 1q23.3, respectively, inhibited cell proliferation. We also found that overexpression of MCL1 or DEDD increased anchorage independence growth in vitro and increased experimental metastasis in vivo in the nonamplified urothelial carcinoma cell line, RT112. The expression of MCL1 confers resistance to a range of apoptosis inducers, while the expression of DEDD led to resistance to TNFα-induced apoptosis. These observations identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinoma. Implications: These studies identify MCL1 and DEDD as genes that contribute to aggressive urothelial carcinomas.

Targeting HER2 with antibodies or small molecule inhibitors in HER2-positive breast cancer leads to improved survival, but resistance is a common clinical problem. To uncover novel mechanisms of resistance to anti-HER2 therapy in breast cancer, we performed a kinase open reading frame screen to identify genes that rescue HER2-amplified breast cancer cells from HER2 inhibition or suppression. In addition to multiple members of the MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase) signaling pathways, we discovered that expression of the survival kinases PRKACA and PIM1 rescued cells from anti-HER2 therapy. Furthermore, we observed elevated PRKACA expression in trastuzumab-resistant breast cancer samples, indicating that this pathway is activated in breast cancers that are clinically resistant to trastuzumab-containing therapy. We found that neither PRKACA nor PIM1 restored MAPK or PI3K activation after lapatinib or trastuzumab treatment, but rather inactivated the pro-apoptotic protein BAD, the BCl-2-associated death promoter, thereby permitting survival signaling through BCL-XL. Pharmacological blockade of BCL-XL/BCL-2 partially abrogated the rescue effects conferred by PRKACA and PIM1, and sensitized cells to lapatinib treatment. These observations suggest that combined targeting of HER2 and the BCL-XL/BCL-2 anti-apoptotic pathway may increase responses to anti-HER2 therapy in breast cancer and decrease the emergence of resistant disease.

Amplification and/or overexpression of the receptor tyrosine kinase HER2 occurs in 20-25% of breast cancers, and is associated with poor prognosis. Targeting of HER2 with drugs such as trastuzumab, lapatinib, or pertuzumab has led to clinical benefit in patients with both metastatic and early-stage HER2-amplified breast cancer. However, resistance and disease progression always occurs in patients with metastatic disease, and many patients with early-stage breast cancer experience recurrences despite adjuvant anti-HER2 therapy. As such, understanding the mechanisms of resistance to anti-HER2 therapy has important clinical implications. Recent studies have identified mutations in PIK3CA, the gene encoding the catalytic subunit of Phosphatidylinositol 3 kinase (PI3K), as one mechanism of resistance to trastuzumab. However, such mutations are present in only a fraction of trastuzumab-resistant breast cancers. We therefore sought to uncover novel mechanisms of resistance to anti-HER2 therapy through an unbiased screen for kinases and kinase-related molecules that are able to rescue HER2-amplified breast cancer cells from HER2 inhibition. We utilized a library of nearly 600 lentivirally-delivered open reading frames (ORFs) to constitutively express the coding sequence of each molecule individually in HER2-amplified BT474 breast cancer cells in arrayed high-throughput format. We conducted two parallel screens for the ability of each of these molecules to rescue cells from anti-HER2 therapy: one in which we treated the cells with a lapatinib-like drug that inhibits the kinase activity of HER2 and EGFR, and one in which we lentivirally delivered a short hairpin RNA that suppresses expression of HER2. We identified those ORFs that restored viability of BT474 cells to greater than two standard deviations above the median of all ORFs in each screen. Multiple members of the MAPK and PI3K signaling pathways scored in both screens, serving to validate the approach. In addition, the survival kinases PIM1 and PRKACA scored robustly. Mechanistic studies suggest that these kinases may confer resistance by restoring the phosphorylation of, and thereby inactivating, the pro-apoptotic protein BAD. Consistent with this finding, overexpression of Bcl-xl, which is inhibited by BAD, also conferred resistance to lapatinib in HER2-amplified breast cancer cells. Furthermore, pharmacological blockade of Bcl-xl and Bcl-2 with ABT-263 enhanced lapatinib-induced killing of HER2-amplified breast cancer cells in vitro, and partially abrogated the rescue conferred by both PRKACA and PIM1. These findings suggest that combined inhibition of HER2 and the anti-apoptotic molecules Bcl-xl and Bcl-2 could enhance tumor cell eradication and prevent or delay the emergence of resistant disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-01.

In prostate cancer, the development of castration resistance is pivotal in progression to aggressive disease. However, understanding of the pathways involved remains incomplete. In this study, we performed a high-throughput genetic screen to identify kinases that enable tumor formation by androgen-dependent prostate epithelial (LHSR-AR) cells under androgen-deprived conditions. In addition to the identification of known mediators of castration resistance, which served to validate the screen, we identified a mitotic-related serine/threonine kinase, NEK6, as a mediator of androgen-independent tumor growth. NEK6 was overexpressed in a subset of human prostate cancers. Silencing NEK6 in castration-resistant cancer cells was sufficient to restore sensitivity to castration in a mouse xenograft model system. Tumors in which castration resistance was conferred by NEK6 were predominantly squamous in histology with no evidence of AR signaling. Gene expression profiling suggested that NEK6 overexpression stimulated cytoskeletal, differentiation, and immune signaling pathways and maintained gene expression patterns normally decreased by castration. Phosphoproteome profiling revealed the transcription factor FOXJ2 as a novel NEK6 substrate, with FOXJ2 phosphorylation associated with increased expression of newly identified NEK6 transcriptional targets. Overall, our studies establish NEK6 signaling as a central mechanism mediating castration-resistant prostate cancer. Cancer Res; 77(3); 753–65. ©2016 AACR.

Natural killer (NK) cells are primary effectors of innate immunity directed against transformed tumor cells. In response, tumor cells have developed mechanisms to evade NK cell-mediated lysis through molecular mechanisms that are not well understood. In the present study, we used a lentiviral shRNA library targeting more than 1,000 human genes to identify 83 genes that promote target cell resistance to human NK cell-mediated killing. Many of the genes identified in this genetic screen belong to common signaling pathways; however, none of them have previously been known to modulate susceptibility of human tumor cells to immunologic destruction. Gene silencing of two members of the JAK family (JAK1 and JAK2) increased the susceptibility of a variety of tumor cell types to NK-mediated lysis and induced increased secretion of IFN-γ by NK cells. Treatment of tumor cells with JAK inhibitors also increased susceptibility to NK cell activity. These findings may have important clinical implications and suggest that small molecule inhibitors of tyrosine kinases being developed as therapeutic antitumor agents may also have significant immunologic effects in vivo.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults. Long-term survival of patients with AML has changed little over the past decade, necessitating the identification and validation of new AML targets. Integration of genomic approaches with small-molecule and genetically based high-throughput screening holds the promise of improved discovery of candidate targets for cancer therapy. Here, we identified a role for glycogen synthase kinase 3α (GSK-3α) in AML by performing 2 independent small-molecule library screens and an shRNA screen for perturbations that induced a differentiation expression signature in AML cells. GSK-3 is a serine-threonine kinase involved in diverse cellular processes, including differentiation, signal transduction, cell cycle regulation, and proliferation. We demonstrated that specific loss of GSK-3α induced differentiation in AML by multiple measurements, including induction of gene expression signatures, morphological changes, and cell surface markers consistent with myeloid maturation. GSK-3α-specific suppression also led to impaired growth and proliferation in vitro, induction of apoptosis, loss of colony formation in methylcellulose, and anti-AML activity in vivo. Although the role of GSK-3β has been well studied in cancer development, these studies support a role for GSK-3α in AML.

SummaryMYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.PaperFlick

Inhibition of CDK4/6 has emerged as an effective therapy in human cancer. Palbociclib, the first selective CDK4/6 inhibitor (CDK4/6i), is FDA-approved for treatment of metastatic breast cancer, and abemaciclib has been fast tracked for approval as a front line breast cancer treatment. Recent evidence in mouse models of breast cancer further indicates that CDK4/6 inhibition triggers anti-tumor immunity, but the precise tumor intrinsic mechanism that mediates CDK4/6 inhibition for a clinical response remains to be defined. To address this, we inhibited CDK4/6 with palbociclib in sequential combination with immunomodulary drugs (IMiD)s in primary bone marrow myeloma cells (BMMCs), using normal bone marrow plasma cells as a control. IMiDs, lenalidomide (Len) and pomalidomide (Pom), are standard of care for multiple myeloma (MM) and lymphoma. Each rarely achieves complete remission despite a high overall response rate and durability. However, each markedly potentiates the clinical efficacy of diverse partners by unknown mechanisms. Cereblon (CRBN), a substrate receptor of the CUL4-ROC1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase, is requited for IMiD's anti-tumor activity. Binding of Len or Pom to CRBN accelerates the recruitment of transcription factors IKZF1/3 to CRL4CRBN for ubiquitination and degradation in MM cells. One of the targets of IKZF1/3 is IRF4, which is required for survival of MM cells and reduced after exposure to Len or Pom. Moreover, MEIS2, a homeobox transcription factor was identified to be an substrate of CRBN in crystal structure and by biochemical screen, implicating a role for MEIS2 in modulates IMiD's anti-tumor activity. However, evidence for MEIS2 expression in myeloma cells is lacking. Previously, we have shown that prolonged early G1 arrest (pG1) induced by sustained CDK4/6 inhibition reprogrammed cancer cells for vulnerability to diverse clinically relevant agents ex vivo and in animal models. To address if CDK4 inhibition reprograms MM cells for IMiD vulnerability, first we show that the intrinsic IMiD sensitivity in primary BMMCs ex vivo is a major determinant for the clinical response to IMiD therapy in myeloma patients. Second, we discovered that MEIS2 is aberrantly expressed and associated with CRBN in BMMCs. Third, MEIS2 is regulated by the cell cycle in primary MM cells and required for the survival of MM cells in part by maintaining IRF4 expression. Finally, CDK4/6 inhibition liberates CRBN from association with MEIS2 in pG1. This rapidly accelerates Len-mediated ubiquitination and degradation of IKZF1/3, loss of IRF4, de-repression of IRF7 and induction of interferon (IFN) responsive genes that culminated in TRAIL-mediated apoptosis. Dissociation of MEIS2 from CRBN thus represents a noel mechanism by which CDK4/6 inhibition reprograms myeloma for vulnerability to IMiDs through induction anti-tumor IFN response. Citation Format: Xiangao Huang, David Jayabalan, Maurizio Di Liberto, Zhengming Chen, Anna Schinzel, Scott A. Ely, Adriana Rossi, Morton Coleman, Joseph M. Lane, William C. Hahn, Tomer M. Mark, Ruben Niesvizky, Selina Chen-Kiang. CDK4/6 inhibition triggers dissociation of MEIS2 from CRL4CRBN E3 ligase to induce interferon response and reprogram myeloma for IMiD vulnerability [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 2300.

SummaryOvarian cancer is a leading cause of death from gynecologic malignancies. Treatment for advanced-stage disease remains limited and, to date, targeted therapies have been incompletely explored. By systematically suppressing each human tyrosine kinase in ovarian cancer cell lines by RNAi, we found that an autocrine signal-transducing loop involving NRG1 and activated ErbB3 operates in a subset of primary ovarian cancers and ovarian cancer cell lines. Perturbation of this circuit with ErbB3-directed RNAi decreased cell growth in three-dimensional culture and resulted in decreased disease progression and prolonged survival in a xenograft mouse model of ovarian cancer. Furthermore, a monoclonal ErbB3-directed antibody (MM-121) also significantly inhibited tumor growth in vivo. These findings identify ErbB3 as a potential therapeutic target in ovarian cancer.

The mechanisms by which cytokine signals prevent the activation and mitochondrial targeting of the proapoptotic protein Bax are unclear. Here we show, using primary human eosinophils, that in the absence of the prosurvival cytokines granulocyte-macrophage colony-stimulating factor and interleukin 5, Bax spontaneously underwent activation and initiated mitochondrial disruption. Inhibition of Bax resulted in less eosinophil apoptosis, even in the absence of cytokines. Granulocyte-macrophage colony-stimulating factor induced activation of the kinase Erk1/2, which phosphorylated Thr167 of Bax; this facilitated new interaction of Bax with the prolyl isomerase Pin1. Blockade of Pin1 led to cleavage and mitochondrial translocation of Bax and caspase activation, regardless of the presence of cytokines. Our findings indicate that Pin1 is a key mediator of prosurvival signaling and is a regulator of Bax function.

Current therapies for sarcomas are often inadequate. This study sought to identify actionable gene targets by selective targeting of the molecular networks that support sarcoma cell proliferation. Silencing of asparagine synthetase (ASNS), an amidotransferase that converts aspartate into asparagine, produced the strongest inhibitory effect on sarcoma growth in a functional genomic screen of mouse sarcomas generated by oncogenic Kras and disruption of Cdkn2a. ASNS silencing in mouse and human sarcoma cell lines reduced the percentage of S phase cells and impeded new polypeptide synthesis. These effects of ASNS silencing were reversed by exogenous supplementation with asparagine. Also, asparagine depletion via the ASNS inhibitor amino sulfoximine 5 (AS5) or asparaginase inhibited mouse and human sarcoma growth in vitro, and genetic silencing of ASNS in mouse sarcoma cells combined with depletion of plasma asparagine inhibited tumor growth in vivo. Asparagine reliance of sarcoma cells may represent a metabolic vulnerability with potential anti-sarcoma therapeutic value. DOI: http://dx.doi.org/10.7554/eLife.09436.001, eLife digest Sarcoma is a type of cancer that forms in the connective tissues of the body, such as bone, cartilage, muscle and fat. Usually, treatment involves surgical removal of the tumor and/or radiation to kill the tumor cells. However, if sarcomas spread to other parts of the body, the treatment options are limited. Genetic studies have revealed several genetic changes that contribute to the formation of sarcomas. Many sarcomas have a mutation in a gene that encodes a protein called Ras. In 2011, researchers found that injecting Ras mutant muscle cells into the muscles of mice could lead to the formation of sarcomas. Next, the researchers compared gene expression in the mouse sarcoma cells with gene expression in normal mouse muscle cells and found that certain genes appeared to be more highly expressed in the sarcoma cells. These genes were also hyperactive in human sarcoma cells and may promote the growth of sarcomas carrying mutant forms of Ras. Now, Hettmer et al. – including some of the same researchers involved in the earlier work – show that targeting one of these hyperactive genes can slow sarcoma growth. The experiments made use of a technique called ribonucleic acid interference (or RNAi for short) to specifically switch off the expression of the hyperactive genes and then observed how this affected sarcoma growth. Hettmer et al. found that blocking the expression of one particular gene, which encodes an enzyme called asparagine synthetase, slowed down the growth of the sarcoma the most. Asparagine synthetase makes the amino acid asparagine, which is needed to make proteins in cells. Further experiments showed that reducing the amount of asparagine in human and mouse sarcoma cells slowed down the growth of these cells. A drug that lowers the amount of asparagine in cells is already used to treat some blood cancers. Hettmer et al.’s findings suggest that drugs that alter the availability of asparagine in the body might also be useful to treat sarcomas with mutant forms of Ras. DOI: http://dx.doi.org/10.7554/eLife.09436.002

Accumulation of misfolded protein in the endoplasmic reticulum (ER) triggers an adaptive stress response-termed the unfolded protein response (UPR)-mediated by the ER transmembrane protein kinase and endoribonuclease inositol-requiring enzyme-1alpha (IRE1alpha). We investigated UPR signaling events in mice in the absence of the proapoptotic BCL-2 family members BAX and BAK [double knockout (DKO)]. DKO mice responded abnormally to tunicamycin-induced ER stress in the liver, with extensive tissue damage and decreased expression of the IRE1 substrate X-box-binding protein 1 and its target genes. ER-stressed DKO cells showed deficient IRE1alpha signaling. BAX and BAK formed a protein complex with the cytosolic domain of IRE1alpha that was essential for IRE1alpha activation. Thus, BAX and BAK function at the ER membrane to activate IRE1alpha signaling and to provide a physical link between members of the core apoptotic pathway and the UPR.

Mitochondrial permeability transition (PT) is a phenomenon induced by high levels of matrix calcium and is characterized by the opening of the PT pore (PTP). Activation of the PTP results in loss of mitochondrial membrane potential, expansion of the matrix, and rupture of the mitochondrial outer membrane. Consequently, PT has been implicated in both apoptotic and necrotic cell death. Cyclophilin D (CypD) appears to be a critical component of the PTP. To investigate the role of CypD in cell death, we created a CypD-deficient mouse. In vitro , CypD-deficient mitochondria showed an increased capacity to retain calcium and were no longer susceptible to PT induced by the addition of calcium. CypD-deficient primary mouse embryonic fibroblasts (MEFs) were as susceptible to classical apoptotic stimuli as the WT, suggesting that CypD is not a central component of cell death in response to these specific death stimuli. However, CypD-deficient MEFs were significantly less susceptible than their WT counterparts to cell death induced by hydrogen peroxide, implicating CypD in oxidative stress-induced cell death. Importantly, CypD-deficient mice displayed a dramatic reduction in brain infarct size after acute middle cerebral artery occlusion and reperfusion, strongly supporting an essential role for CypD in an ischemic injury model in which calcium overload and oxidative stress have been implicated.

High-grade serous ovarian carcinoma (HGSOC) is the most common and aggressive form of epithelial ovarian cancer, for which few targeted therapies exist. To search for new therapeutic target proteins, we performed an in vivo shRNA screen using an established human HGSOC cell line growing either subcutaneously or intraperitoneally in immunocompromised mice. We identified genes previously implicated in ovarian cancer such as AURKA1, ERBB3, CDK2, and mTOR, as well as several novel candidates including BRD4, VRK1, and GALK2. We confirmed, using both genetic and pharmacologic approaches, that the activity of BRD4, an epigenetic transcription modulator, is necessary for proliferation/survival of both an established human ovarian cancer cell line (OVCAR8) and a subset of primary serous ovarian cancer cell strains (DFs). Among the DFs tested, the strains sensitive to BRD4 inhibition revealed elevated expression of either MYCN or c-MYC, with MYCN expression correlating closely with JQ1 sensitivity. Accordingly, primary human xenografts derived from high-MYCN or c-MYC strains exhibited sensitivity to BRD4 inhibition. These data suggest that BRD4 inhibition represents a new therapeutic approach for MYC-overexpressing HGSOCs.

Although the roles of mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling in KRAS-driven tumorigenesis are well established, KRAS activates additional pathways required for tumor maintenance, the inhibition of which are likely to be necessary for effective KRAS-directed therapy. Here, we show that the IκB kinase (IKK)–related kinases Tank-binding kinase-1 (TBK1) and IKKϵ promote KRAS-driven tumorigenesis by regulating autocrine CCL5 and interleukin (IL)-6 and identify CYT387 as a potent JAK/TBK1/IKKϵ inhibitor. CYT387 treatment ablates RAS-associated cytokine signaling and impairs Kras-driven murine lung cancer growth. Combined CYT387 treatment and MAPK pathway inhibition induces regression of aggressive murine lung adenocarcinomas driven by Kras mutation and p53 loss. These observations reveal that TBK1/IKKϵ promote tumor survival by activating CCL5 and IL-6 and identify concurrent inhibition of TBK1/IKKϵ, Janus-activated kinase (JAK), and MEK signaling as an effective approach to inhibit the actions of oncogenic KRAS. Significance: In addition to activating MAPK and PI3K, oncogenic KRAS engages cytokine signaling to promote tumorigenesis. CYT387, originally described as a selective JAK inhibitor, is also a potent TBK/IKKϵ inhibitor that uniquely disrupts a cytokine circuit involving CCL5, IL-6, and STAT3. The efficacy of CYT387-based treatment in murine Kras-driven lung cancer models uncovers a novel therapeutic approach for these refractory tumors with immediate translational implications. Cancer Discov; 4(4); 452–65. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 377

Down syndrome confers a 20-fold increased risk of B cell acute lymphoblastic leukemia (B-ALL), and polysomy 21 is the most frequent somatic aneuploidy among all B-ALLs. Yet the mechanistic links between chromosome 21 triplication and B-ALL remain undefined. Here we show that germline triplication of only 31 genes orthologous to human chromosome 21q22 confers mouse progenitor B cell self renewal in vitro, maturation defects in vivo and B-ALL with either the BCR-ABL fusion protein or CRLF2 with activated JAK2. Chromosome 21q22 triplication suppresses histone H3 Lys27 trimethylation (H3K27me3) in progenitor B cells and B-ALLs, and 'bivalent' genes with both H3K27me3 and H3K4me3 at their promoters in wild-type progenitor B cells are preferentially overexpressed in triplicated cells. Human B-ALLs with polysomy 21 are distinguished by their overexpression of genes marked with H3K27me3 in multiple cell types. Overexpression of HMGN1, a nucleosome remodeling protein encoded on chromosome 21q22 (refs. 3,4,5), suppresses H3K27me3 and promotes both B cell proliferation in vitro and B-ALL in vivo.

Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling., National Institutes of Health (U.S.) (Grant K99 CA169512), National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051), Virginia and D.K. Ludwig Fund for Cancer Research, American Association for Cancer Research, Howard Hughes Medical Institute, Leukemia & Lymphoma Society of America, United States. Department of Defense (Fellowship W81XWH-10-1-0062)

3q26 is frequently amplified in several cancer types with a common amplified region containing 20 genes. To identify cancer driver genes in this region, we interrogated the function of each of these genes by loss- and gain-of-function genetic screens. Specifically, we found that TLOC1 (SEC62) was selectively required for the proliferation of cell lines with 3q26 amplification. Increased TLOC1 expression induced anchorage-independent growth, and a second 3q26 gene, SKIL (SNON), facilitated cell invasion in immortalized human mammary epithelial cells. Expression of both TLOC1 and SKIL induced subcutaneous tumor growth. Proteomic studies showed that TLOC1 binds to DDX3X, which is essential for TLOC1-induced transformation and affected protein translation. SKIL induced invasion through upregulation of SLUG (SNAI2) expression. Together, these studies identify TLOC1 and SKIL as driver genes at 3q26 and more broadly suggest that cooperating genes may be coamplified in other regions with somatic copy number gain. Significance: These studies identify TLOC1 and SKIL as driver genes in 3q26. These observations provide evidence that regions of somatic copy number gain may harbor cooperating genes of different but complementary functions. Cancer Discov; 3(9); 1044–57. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 953

SummaryWnt/β-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for cancer initiation and progression. To decipher the role of β-catenin in transformation, we classified β-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that β-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with β-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of β-catenin-dependent cancers in both cell lines and animal models. These observations define a β-catenin-YAP1-TBX5 complex essential to the transformation and survival of β-catenin-driven cancers.

The NKX2-1 transcription factor, a regulator of normal lung development, is the most significantly amplified gene in human lung adenocarcinoma. To study the transcriptional impact of NKX2-1 amplification, we generated an expression signature associated with NKX2-1 amplification in human lung adenocarcinoma and analyzed DNA-binding sites of NKX2-1 by genome-wide chromatin immunoprecipitation. Integration of these expression and cistromic analyses identified LMO3, itself encoding a transcription regulator, as a candidate direct transcriptional target of NKX2-1. Further cistromic and overexpression analyses indicated that NKX2-1 can cooperate with the forkhead box transcription factor FOXA1 to regulate LMO3 gene expression. RNAi analysis of NKX2-1-amplified cells compared with nonamplified cells demonstrated that LMO3 mediates cell survival downstream from NKX2-1. Our findings provide new insight into the transcriptional regulatory network of NKX2-1 and suggest that LMO3 is a transcriptional signal transducer in NKX2-1-amplified lung adenocarcinomas., National Institutes of Health (U.S.) (NIH/NCI T32 Institutional Training Program fellowship (5T32CA009361-28)), National Cancer Institute (U.S.) (grant 5R01CA109038), National Cancer Institute (U.S.) (grant 5P20CA90578)

One of the central goals of human genetics is to discover the genes and pathways driving human traits. To date, most of the common risk alleles discovered through genome-wide association studies (GWAS) map to nonprotein-coding regions. Because of our relatively poorer understanding of this part of the genome, the functional consequences of trait-associated variants pose a considerable challenge. To identify the genes through which risk loci act, we hypothesized that the risk variants are regulatory elements. For each of 12 known risk polymorphisms, we evaluated the correlation between risk allele status and transcript abundance for all annotated protein-coding transcripts within a 1-Mb interval. A total of 103 transcripts were evaluated in 662 prostate tissue samples [normal ( n = 407) and tumor ( n = 255)] from 483 individuals [European Americans ( n = 233), Japanese ( n = 127), and African Americans ( n = 123)]. In a pooled analysis, 4 of the 12 risk variants were strongly associated with five transcripts ( NUDT11 , MSMB , NCOA4 , SLC22A3 , and HNF1B ) in histologically normal tissue ( P ≤ 0.001). Although associations were also observed in tumor tissue, they tended to be more attenuated. Previously, we showed that MSMB and NCOA4 participate in prostate cancer pathogenesis. Suppressing the expression of NUDT11 , SLC22A3 , and HNF1B influences cellular phenotypes associated with tumor-related properties in prostate cancer cells. Taken together, the data suggest that these transcripts contribute to prostate cancer pathogenesis.