Wouter Nijkamp

A large-scale RNAi screen in human cells identifies new components of the p53 pathway

RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function genetic screens in lower organisms and can greatly facilitate the identification of components of cellular signalling pathways. In mammalian cells, such screens have been hampered by a lack of suitable tools that can be used on a large scale. We and others have recently developed expression vectors to direct the synthesis of short hairpin RNAs (shRNAs) that act as short interfering RNA (siRNA)-like molecules to stably suppress gene expression. Here we report the construction of a set of retroviral vectors encoding 23,742 distinct shRNAs, which target 7,914 different human genes for suppression. We use this RNAi library in human cells to identify one known and five new modulators of p53-dependent proliferation arrest. Suppression of these genes confers resistance to both p53-dependent and p19ARF-dependent proliferation arrest, and abolishes a DNA-damage-induced G1 cell-cycle arrest. Furthermore, we describe siRNA bar-code screens to rapidly identify individual siRNA vectors associated with a specific phenotype. These new tools will greatly facilitate large-scale loss-of-function genetic screens in mammalian cells.

Phosphatidylinositol 3-Kinase Hyperactivation Results in Lapatinib Resistance that Is Reversed by the mTOR/Phosphatidylinositol 3-Kinase Inhibitor NVP-BEZ235

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 short hairpin RNA screen to identify novel modulators of resistance to lapatinib, a recently approved anti-HER2 tyrosine kinase inhibitor. Here, we have identified the tumor suppressor PTEN as a modulator of lapatinib sensitivity in vitro and in vivo. In addition, we show 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 phosphatidylinositol 3-kinase (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.

MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling

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 MED12(KD) cells, suggesting a strategy to treat drug-resistant tumors that have lost MED12.

NF1 Is a Tumor Suppressor in Neuroblastoma that Determines Retinoic Acid Response and Disease Outcome

Retinoic acid (RA) induces differentiation of neuroblastoma cells in vitro and is used with variable success to treat aggressive forms of this disease. This variability in clinical response to RA is enigmatic, as no mutations in components of the RA signaling cascade have been found. Using a large-scale RNAi genetic screen, we identify crosstalk between the tumor suppressor NF1 and retinoic acid-induced differentiation in neuroblastoma. Loss of NF1 activates RAS-MEK signaling, which in turn represses ZNF423, a critical transcriptional coactivator of the retinoic acid receptors. Neuroblastomas with low levels of both NF1 and ZNF423 have extremely poor outcome. We find NF1 mutations in neuroblastoma cell lines and in primary tumors. Inhibition of MEK signaling downstream of NF1 restores responsiveness to RA, suggesting a therapeutic strategy to overcome RA resistance in NF1-deficient neuroblastomas.

ZNF423 Is Critically Required for Retinoic Acid-Induced Differentiation and Is a Marker of Neuroblastoma Outcome

Retinoids play key roles in differentiation, growth arrest, and apoptosis and are increasingly being used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Here, 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 RARalpha/RXRalpha nuclear receptor complex and is essential for transactivation in response to retinoids. Downregulation 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 in neuroblastoma patients.

Telomerase independent regulation of ATR by human telomerase RNA

The human telomerase RNA (hTR), together with the telomerase reverse transcriptase, hTERT, constitute the core components of telomerase that is essential for telomere maintenance. While hTR is ubiquitously expressed, hTERT is normally restricted to germ cells and certain stem cells, but both are often deregulated during tumorigenesis. Here, we investigated the effects of changes in hTR cellular levels. Surprisingly, while inhibition of hTR expression triggers a rapid, telomerase-independent, growth arrest associated with p53 and CHK1 activation, its increased expression neutralizes activation of these pathways in response to genotoxic stress. These hTR effects are mediated through ATR and are sufficiently strong to impair ATR-mediated DNA-damage checkpoint responses. Furthermore, in response to low UV radiation, which activates ATR, endogenous hTR levels increase irrespective of telomerase status. Thus, we uncovered a novel, telomerase-independent, function of hTR that restrains ATR activity and participates in the recovery of cells from UV radiation.

SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer

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.

The Corepressor CTBP2 Is a Coactivator of Retinoic Acid Receptor/Retinoid X Receptor in Retinoic Acid Signaling

ABSTRACT 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.

Abstract LB-240: SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Recurrent inactivating mutations in components of SWI/SNF chromatin-remodelling complexes have been identified across cancer types, supporting their roles as tumour suppressors in modulating oncogenic signalling 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 signalling and suggest targeted treatment options for SMARCE1 deficient tumours. ACKNOWLEDGEMENTS: *Equal contribution. #Corresponding authors. This work was supported by grants from Canadian Institutes of Health Research (CIHR) grant MOP-130540 (S.H.), Canada Research Chair (CRC), the European Research Council (ERC), the Dutch Cancer Society (KWF), the EU COLTHERES project and grants by the Netherlands Organization for Scientific Research (NWO) to the Cancer Genomics Center Netherlands (CGC.NL). Citation Format: Andreas Papadakis*, Chong Sun*, Theo Knijnenburg, Yibo Xue, Wipawadee Grernrum, Michael Holzel, Wouter Nijkamp, Lodewyk Wessels, Roderick Beijersbergen, Rene Bernards#, Sidong Huang#. SMARCE1 suppresses EGFR expression and controls responses to MET and ALK inhibitors in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-240. doi:10.1158/1538-7445.AM2015-LB-240

Abstract A30: NF1 is a tumor suppressor in neuroblastoma that determines retinoic acid response and disease outcome

Neuroblastoma is a malignancy of early childhood that derives from the developing neural crest and accounts for 15% of all paediatric oncology deaths. The vitamin A derivative retinoic acid (RA) controls neural development and promotes differentiation, cell cycle arrest and apoptosis of neuronal and neuroblastoma cells. These diverse effects of RA are exerted primarily through the ability to differentially regulate gene expression mediated by the retinoic acid receptors (RARs). RA is used in the clinic to treat high-risk neuroblastoma patients, however with variable success. This variability in clinical response to RA is enigmatic as no mutations in components of the RA signaling cascade have been found. By using large-scale RNAi-based genetic screens, we aimed to identify predictive biomarkers for RA sensitivity in neuroblastoma. We found an unexpected crosstalk between the tumor suppressor neurofibromin (NF1) and RA signaling. NF1 is a negative regulator of RAS signaling that promotes conversion of the active GTP bound form into the inactive GDP bound form of RAS. Activation of RAS-MEK signaling by loss of NF1 repressed ZNF423, a critical transcriptional co-activator of the retinoic acid receptors, and blocked RA induced differentiation and target gene activation. Restoration of ZNF423 levels resensitized NF1 knockdown cells to the inhibitory effect of RA. Moreover, ZNF423 and NF1 expression predicted sensitivity to RA in a large panel of neuroblastoma cell lines. Neuroblastomas with low levels of both NF1 and ZNF423 have extremely poor outcome. We found NF1 mutations in neuroblastoma cell lines and also in primary tumors. Inhibition of MEK signaling downstream of NF1 restored responsiveness to RA, suggesting a therapeutic strategy to overcome RA resistance in NF1 deficient neuroblastomas. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A30