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1.  Intrinsic resistance to PIM kinase inhibition in AML through p38α-mediated feedback activation of mTOR signaling 
Oncotarget  2016;7(25):37407-37419.
Although conventional therapies for acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) are effective in inducing remission, many patients relapse upon treatment. Hence, there is an urgent need for novel therapies. PIM kinases are often overexpressed in AML and DLBCL and are therefore an attractive therapeutic target. However, in vitro experiments have demonstrated that intrinsic resistance to PIM inhibition is common. It is therefore likely that only a minority of patients will benefit from single agent PIM inhibitor treatment. In this study, we performed an shRNA-based genetic screen to identify kinases whose suppression is synergistic with PIM inhibition. Here, we report that suppression of p38α (MAPK14) is synthetic lethal with the PIM kinase inhibitor AZD1208. PIM inhibition elevates reactive oxygen species (ROS) levels, which subsequently activates p38α and downstream AKT/mTOR signaling. We found that p38α inhibitors sensitize hematological tumor cell lines to AZD1208 treatment in vitro and in vivo. These results were validated in ex vivo patient-derived AML cells. Our findings provide mechanistic and translational evidence supporting the rationale to test a combination of p38α and PIM inhibitors in clinical trials for AML and DLBCL.
PMCID: PMC5122321  PMID: 27270648
AML; PIM; AZD1208; p38; resistance
2.  Identification of F-box only protein 7 as a negative regulator of NF-kappaB signalling 
The nuclear factor κB (NF-κB) signalling pathway controls important cellular events such as cell proliferation, differentiation, apoptosis and immune responses. Pathway activation occurs rapidly upon TNFα stimulation and is highly dependent on ubiquitination events. Using cytoplasmic to nuclear translocation of the NF-κB transcription factor family member p65 as a read-out, we screened a synthetic siRNA library targeting enzymes involved in ubiquitin conjugation and de-conjugation for modifiers of regulatory ubiquitination events in NF-κB signalling. We identified F-box protein only 7 (FBXO7), a component of Skp, Cullin, F-box (SCF)-ubiquitin ligase complexes, as a negative regulator of NF-κB signalling. F-box protein only 7 binds to, and mediates ubiquitin conjugation to cIAP1 and TRAF2, resulting in decreased RIP1 ubiquitination and lowered NF-κB signalling activity.
PMCID: PMC3822984  PMID: 22212761
FBXO7; NF-κB signalling; RNA interference; siRNA; regulatory ubiquitination; cIAP1
3.  SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer 
Cell Research  2015;25(4):445-458.
Recurrent inactivating mutations in components of SWI/SNF chromatin-remodeling complexes have been identified across cancer types, supporting their roles as tumor suppressors in modulating oncogenic signaling pathways. We report here that SMARCE1 loss induces EGFR expression and confers resistance to MET and ALK inhibitors in non-small cell lung cancers (NSCLCs). We found that SMARCE1 binds to regulatory regions of the EGFR locus and suppresses EGFR transcription in part through regulating expression of Polycomb Repressive Complex component CBX2. Addition of the EGFR inhibitor gefitinib restores the sensitivity of SMARCE1-knockdown cells to MET and ALK inhibitors in NSCLCs. Our findings link SMARCE1 to EGFR oncogenic signaling and suggest targeted treatment options for SMARCE1-deficient tumors.
PMCID: PMC4387553  PMID: 25656847
EGFR signaling; drug resistance; RNAi screening; SMARCE1; SWI/SNF
4.  Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference 
Nucleic Acids Research  2015;43(7):3407-3419.
The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.
PMCID: PMC4402539  PMID: 25800748
5.  The Good, the Bad, and the Ugly: in search of gold standards for assessing functional genetic screen quality 
Molecular Systems Biology  2014;10(7):738.
Variable screen quality, off-target effects, and unclear false discovery rates often hamper large-scale functional genomic screens in mammalian cells. Hart et al (2014) introduce gold standard reference sets of essential and non-essential genes, aiming at standardizing the analysis of genome-wide screens. This work provides a framework to compare both the quality and analysis methods of functional genetic screens.
PMCID: PMC4299495  PMID: 24987117
6.  VAV3 mediates resistance to breast cancer endocrine therapy 
Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process.
A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA–mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression.
The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10−4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy.
This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.
PMCID: PMC4076632  PMID: 24886537
7.  The Corepressor CTBP2 Is a Coactivator of Retinoic Acid Receptor/Retinoid X Receptor in Retinoic Acid Signaling 
Molecular and Cellular Biology  2013;33(16):3343-3353.
Retinoids play key roles in development, differentiation, and homeostasis through regulation of specific target genes by the retinoic acid receptor/retinoid X receptor (RAR/RXR) nuclear receptor complex. Corepressors and coactivators contribute to its transcriptional control by creating the appropriate chromatin environment, but the precise composition of these nuclear receptor complexes remains to be elucidated. Using an RNA interference-based genetic screen in mouse F9 cells, we identified the transcriptional corepressor CTBP2 (C-terminal binding protein 2) as a coactivator critically required for retinoic acid (RA)-induced transcription. CTBP2 suppression by RNA interference confers resistance to RA-induced differentiation in diverse murine and human cells. Mechanistically, we find that CTBP2 associates with RAR/RXR at RA target gene promoters and is essential for their transactivation in response to RA. We show that CTBP2 is indispensable to create a chromatin environment conducive for RAR/RXR-mediated transcription by recruiting the histone acetyltransferase p300. Our data reveal an unexpected function of the corepressor CTBP2 as a coactivator for RAR/RXR in RA signaling.
PMCID: PMC3753894  PMID: 23775127
8.  MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling 
Cell  2012;151(5):937-950.
Inhibitors of the ALK and EGF receptor tyrosine kinases provoke dramatic but short-lived responses in lung cancers harboring EML4-ALK translocations or activating mutations of EGFR, respectively. We used a large-scale RNAi screen to identify MED12, a component of the transcriptional MEDIATOR complex that is mutated in cancers, as a determinant of response to ALK and EGFR inhibitors. MED12 is in part cytoplasmic where it negatively regulates TGF-βR2 through physical interaction. MED12 suppression therefore results in activation of TGF-βR signaling, which is both necessary and sufficient for drug resistance. TGF-β signaling causes MEK/ERK activation, and consequently MED12 suppression also confers resistance to MEK and BRAF inhibitors in other cancers. MED12 loss induces an EMT-like phenotype, which is associated with chemotherapy resistance in colon cancer patients and to gefitinib in lung cancer. Inhibition of TGF-βR signaling restores drug responsiveness in MED12KD cells, suggesting a strategy to treat drug-resistant tumors that have lost MED12.
PMCID: PMC3672971  PMID: 23178117
9.  The Histone Demethylase Jarid1b (Kdm5b) Is a Novel Component of the Rb Pathway and Associates with E2f-Target Genes in MEFs during Senescence 
PLoS ONE  2011;6(9):e25235.
Senescence is a robust cell cycle arrest controlled by the p53 and Rb pathways that acts as an important barrier to tumorigenesis. Senescence is associated with profound alterations in gene expression, including stable suppression of E2f-target genes by heterochromatin formation. Some of these changes in chromatin composition are orchestrated by Rb. In complex with E2f, Rb recruits chromatin modifying enzymes to E2f target genes, leading to their transcriptional repression. To identify novel chromatin remodeling enzymes that specifically function in the Rb pathway, we used a functional genetic screening model for bypass of senescence in murine cells. We identified the H3K4-demethylase Jarid1b as novel component of the Rb pathway in this screening model. We find that depletion of Jarid1b phenocopies knockdown of Rb1 and that Jarid1b associates with E2f-target genes during cellular senescence. These results suggest a role for Jarid1b in Rb-mediated repression of cell cycle genes during senescence.
PMCID: PMC3181323  PMID: 21980403
10.  ZNF423 Is Critically Required for Retinoic Acid-Induced Differentiation and Is a Marker of Neuroblastoma Outcome 
Cancer cell  2009;15(4):328-340.
Retinoids play key roles in differentiation, growth arrest and apoptosis and are increasingly used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Using a large-scale RNA interference-based genetic screen we identify ZNF423 (also known as Ebfaz, OAZ or Zfp423) as a component critically required for retinoic acid (RA)-induced differentiation. ZNF423 associates with the RARα/RXRα nuclear receptor complex and is essential for transactivation in response to retinoids. Down-regulation of ZNF423 expression by RNA interference in neuroblastoma cells results in a growth advantage and resistance to RA-induced differentiation, whereas overexpression of ZNF423 leads to growth inhibition and enhanced differentiation. Finally, we show that low ZNF423 expression is associated with poor disease outcome of neuroblastoma patients.
Cancer biomarkers make it possible to foretell cancer outcome (prognosis) or responses to therapy (prediction). Human neuroblastoma is the most common childhood solid tumor with a broad range of clinical outcomes, ranging from spontaneous regression to extremely aggressive disease. We show here that ZNF423 is a prognostic biomarker for human neuroblastoma independent of MYCN amplification. We also establish here a causal role of ZNF423 in RA-induced differentiation and proliferation of neuroblastoma cells. Therefore, ZNF423 may also predict responses to RA-based therapies in the clinic. More generally, our results underscore that the identification of novel components of key signaling pathways using genetic screens can yield biomarkers having clinical utility.
PMCID: PMC2693316  PMID: 19345331
Retinoic acid receptor; differentiation; neuroblastoma; RNAi
11.  Statistical Methods for Analysis of High-Throughput RNA Interference Screens 
Nature methods  2009;6(8):569-575.
RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery and, in both of these areas, large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
PMCID: PMC2789971  PMID: 19644458
12.  PI3K Hyperactivation Results in Lapatinib Resistance that is Reversed by the mTOR/PI3K Inhibitor NVP-BEZ235 
Cancer research  2008;68(22):9221-9230.
Small molecule inhibitors of HER2 are clinically active in women with advanced HER2 positive breast cancer who have progressed on trastuzumab treatment. However, the effectiveness of this class of agents is limited by either primary resistance or acquired resistance. Using an unbiased genetic approach we performed a genome wide loss-of-function shRNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumour suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we demonstrate that two dominant activating mutations in PIK3CA (E545K and H1047R), which are prevalent in breast cancer, also confer resistance to lapatinib. Furthermore, we show that PI3K induced lapatinib resistance can be abrogated through the use of NVP-BEZ235, a dual inhibitor of PI3K/mTOR. Our data show that deregulation of the PI3K pathway, either through loss-of-function mutations in PTEN or dominant activating mutations in PIK3CA, leads to lapatinib resistance which can be effectively reversed by NVP-BEZ235.
PMCID: PMC2587064  PMID: 19010894
Breast cancer; lapatinib; barcode screen; PI3K pathway; PI3K inhibitors
13.  A Large Scale shRNA Barcode Screen Identifies the Circadian Clock Component ARNTL as Putative Regulator of the p53 Tumor Suppressor Pathway 
PLoS ONE  2009;4(3):e4798.
The p53 tumor suppressor gene is mutated in about half of human cancers, but the p53 pathway is thought to be functionally inactivated in the vast majority of cancer. Understanding how tumor cells can become insensitive to p53 activation is therefore of major importance. Using an RNAi-based genetic screen, we have identified three novel genes that regulate p53 function.
We have screened the NKI shRNA library targeting 8,000 human genes to identify modulators of p53 function. Using the shRNA barcode technique we were able to quickly identify active shRNA vectors from a complex mixture. Validation of the screening results indicates that the shRNA barcode technique can reliable identify active shRNA vectors from a complex pool. Using this approach we have identified three genes, ARNTL, RBCK1 and TNIP1, previously unknown to regulate p53 function. Importantly, ARNTL (BMAL1) is an established component of the circadian regulatory network. The latter finding adds to recent observations that link circadian rhythm to the cell cycle and cancer. We show that cells having suppressed ARNTL are unable to arrest upon p53 activation associated with an inability to activate the p53 target gene p21CIP1.
We identified three new regulators of the p53 pathway through a functional genetic screen. The identification of the circadian core component ARNTL strengthens the link between circadian rhythm and cancer.
PMCID: PMC2653142  PMID: 19277210

Results 1-13 (13)