Immunohistochemical detection of PAX-FOXO1 fusion proteins in alveolar rhabdomyosarcoma using breakpoint specific monoclonal antibodies.

Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.

In vitro activity of a G-quadruplex-stabilizing small molecule that synergizes with Navitoclax to induce cytotoxicity in acute myeloid leukemia cells.

BACKGROUND: Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that arise from genomic alterations in the expression of key growth regulatory genes causing cells to assume an undifferentiated state and continue to proliferate. Recent efforts have focused on developing therapies that target specific protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target and thought to be "undrugable" because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A novel technology that circumvents some of these issues is the use of small molecules that stabilize secondary DNA structures present in the promoters of many potential oncogenes and modulate their transcription. METHODS: This study characterizes the in vitro activity of the G-quadruplex-stabilizing small molecule GQC-05 in AML cells. The effect of GQC-05 on three AML cell lines was analyzed using viability and apoptosis assays. GQC-05 has been shown to down-regulate MYC through G-quadruplex stabilization in Burkitt's lymphoma cell lines. MYC expression was evaluated through qPCR and immunoblotting in the three AML cell lines following the treatment of GQC-05. In order to identify other therapeutic agents that potentiate the activity of GQC-05, combination drug screening was performed. The drug combinations were validated using in vitro cytotoxicity assays and compared to other commonly used chemotherapeutic agents. RESULTS: GQC-05 treatment of KG-1a, CMK and TF-1 cells decreased cell viability and resulted in increased DNA damage and apoptosis. Additionally, treatment of KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with a more pronounced effect in KG-1a cells. Combination drug screening identified the Bcl-2/Bcl-XL inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed a synergistic decrease in cell viability of AML cells as determined by Chou-Talalay analysis, and induced more DNA damage, apoptosis, and rapid cytotoxicity. The cytotoxicity induced by GQC-05 and Navitoclax was more potent than that of Navitoclax combined with either cytarabine or doxorubicin. CONCLUSION: These results suggest that the G-quadruplex stabilizing small molecule GQC-05 induces down regulated MYC expression and DNA damage in AML cells. Treatment with both GQC-05 with a Bcl-2/Bcl-XL inhibitor Navitoclax results in increased cytotoxic activity, which is more pronounced than Navitoclax or GQC-05 alone, and more significant than Navitoclax in combination with cytarabine and doxorubicin that are currently being used clinically.

Clinical resistance associated with a novel MAP2K1 mutation in a patient with Langerhans cell histiocytosis.

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment-refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

Disulfiram overcomes bortezomib and cytarabine resistance in Down-syndrome-associated acute myeloid leukemia cells.

BACKGROUND: Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. METHODS: Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. RESULTS: Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright "stem-like" populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the ß5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. CONCLUSIONS: We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL.

Optimization of Transfection Conditions for siRNA Screening.

RNAi screening of mammalian cells is often performed using siRNAs and cationic lipids as transfection reagents. Efficiency of transfection depends on growth characteristics of the cells and the cationic lipid used. With a large selection of cationic lipids available, it can often be difficult to select the optimal lipid and lipid:siRNA (vol:wt) ratio. Here, we describe the process of optimizing siRNA transfection conditions for efficient reverse transfection of mammalian cells using specific positive and negative siRNA controls.

Data Analysis for High-Throughput RNAi Screening.

High-throughput RNA interference (HT-RNAi) screening is an effective technology to help identify important genes and pathways involved in a biological process. Analysis of high-throughput RNAi screening data is a critical part of this technology, and many analysis methods have been described. Here, we summarize the workflow and types of analyses commonly used in high-throughput RNAi screening.

Aurora B kinase is a potent and selective target in MYCN-driven neuroblastoma.

Despite advances in multimodal treatment, neuroblastoma (NB) is often fatal for children with high-risk disease and many survivors need to cope with long-term side effects from high-dose chemotherapy and radiation. To identify new therapeutic targets, we performed an siRNA screen of the druggable genome combined with a small molecule screen of 465 compounds targeting 39 different mechanisms of actions in four NB cell lines. We identified 58 genes as targets, including AURKB, in at least one cell line. In the drug screen, aurora kinase inhibitors (nine molecules) and in particular the AURKB-selective compound, barasertib, were the most discriminatory with regard to sensitivity for MYCN-amplified cell lines. In an expanded panel of ten NB cell lines, those with MYCN-amplification and wild-type TP53 were the most sensitive to low nanomolar concentrations of barasertib. Inhibition of the AURKB kinase activity resulted in decreased phosphorylation of the known target, histone H3, and upregulation of TP53 in MYCN-amplified, TP53 wild-type cells. However, both wild-type and TP53 mutant MYCN-amplified cell lines arrested in G2/M phase upon AURKB inhibition. Additionally, barasertib induced endoreduplication and apoptosis. Treatment of MYCN-amplified/TP53 wild-type neuroblastoma xenografts resulted in profound growth inhibition and tumor regression. Therefore, aurora B kinase inhibition is highly effective in aggressive neuroblastoma and warrants further investigation in clinical trials.

High-throughput RNAi screening for the identification of novel targets.

Gene silencing through RNA interference has provided researchers with an effective way to study gene function. High-throughput RNA interference (HT-RNAi) screening has further permitted researchers to identify functionally relevant mediators of cellular response on a large scale. These screens have greatly expedited the discovery of novel targets and pathway mediators. Here, we describe the methodology for performing HT-RNAi screening of HeLa cells transfected with short interfering RNA (siRNA) libraries in 384-well microplate format. Using this plate format, the HT-RNAi assay can be easily adapted to semi-automated or fully automated platforms. The library siRNA are introduced into the cells through reverse transfection using cationic lipids. HT-RNAi screening for modulators of cell proliferation can be accomplished using a single read out reagent. This type of RNAi screening can be used with most plate-based cellular assays and can be optimized for most cultured cells lines, thus becoming a powerful tool to identify specific gene modulators and targets for drug discovery.

BIRC5 expression is a poor prognostic marker in Ewing sarcoma.

BACKGROUND: BIRC5 (Survivin), an inhibitor of apoptosis protein (IAP), is over-expressed in several human cancers and increased expression is associated with poor prognosis. The goal of the current study was to evaluate the role of BIRC5 in Ewing sarcoma (ES), the second most common pediatric bone sarcoma. PROCEDURE: BIRC5 protein expression was determined in ES cell lines using Western Blot analysis. Functional role of survivin on growth and viability of ES cells was assessed by siRNA knockdown of BIRC5 and by using a small molecule inhibitor YM155. Immunohistochemical analysis for BIRC5 protein was performed on patient tumor samples using an anti-survivin antibody. The degree of BIRC5 protein expression was correlated with clinical parameters and patient outcome. RESULTS: BIRC5 is over-expressed in a panel of ES cell lines. Gene silencing of BIRC5 in the ES cell line TC-71 decreases cell growth by more than 50% for each BIRC5 siRNA construct compared to non-silencing siRNA control constructs. YM155 also reduces ES cell growth and viability with an EC(50) ranging from 2.8 to 6.2 nM. BIRC5 protein is expressed in majority of the ES tumor samples with minimal expression in normal tissue (P < 0.005). Tumors with more than 50% expression are associated with worse overall survival than tumors with less than 50% expression (Hazard Ratio: 6.05; CI: 1.7-21.4; P = 0.04). CONCLUSION: BIRC5 is over-expressed in ES cell lines and tumor samples. Further, it plays an important role in cell growth and viability in vitro. Higher degree of expression in patients is an independent poor prognostic factor.

Negative regulation of NF-κB by the ING4 tumor suppressor in breast cancer.

Nuclear Factor kappa B (NF-κB) is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4) tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227). Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65), an activated form of NF-κB (p = 0.018). Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in breast cancer.

The genomic and proteomic content of cancer cell-derived exosomes.

Exosomes are secreted membrane vesicles that have been proposed as an effective means to detect a variety of disease states, including cancer. The properties of exosomes, including stability in biological fluids, allow for their efficient isolation and make them an ideal vehicle for studies on early disease detection and evaluation. Much data has been collected over recent years regarding the messenger RNA, microRNA, and protein contents of exosomes. In addition, many studies have described the functional role that exosomes play in disease initiation and progression. Tumor cells have been shown to secrete exosomes, often in increased amounts compared to normal cells, and these exosomes can carry the genomic and proteomic signatures characteristic of the tumor cells from which they were derived. While these unique signatures make exosomes ideal for cancer detection, exosomes derived from cancer cells have also been shown to play a functional role in cancer progression. Here, we review the unique genomic and proteomic contents of exosomes originating from cancer cells as well as their functional effects to promote tumor progression.

Foxo3a drives proliferation in anaplastic thyroid carcinoma through transcriptional regulation of cyclin A1: a paradigm shift that impacts current therapeutic strategies.

The Forkhead transcription factor, FoxO3a, is a known suppressor of primary tumor growth through transcriptional regulation of key genes regulating cell cycle arrest and apoptosis. In many types of cancer, in response to growth factor signaling, FoxO3a is phosphorylated by Akt, resulting in its exclusion from the nucleus. Here we show that FoxO3a remains nuclear in anaplastic thyroid carcinoma (ATC). This correlates with lack of Akt phosphorylation at serine473 in ATC cell lines and tissues of ATC patients, providing a potential explanation for nuclear FoxO3a. Mechanistically, nuclear FoxO3a promotes cell cycle progression by transcriptional upregulation of cyclin A1, promoting proliferation of human ATC cells. Silencing FoxO3a with a reverse genetics approach leads to downregulation of CCNA1 mRNA and protein. These combined data suggest an entirely novel function for FoxO3a in ATC promotion by enhancing cell cycle progression and tumor growth through transcriptional upregulation of cyclin A1. This is clinically relevant since we detected highly elevated CCNA1 mRNA and protein levels in tumor tissues of ATC patients. Our data indicate therapeutic inactivation of FoxO3a may lead to attenuation of tumor expansion in ATC. This new paradigm also suggests caution in relation to current dogma focused upon reactivation of FoxO3a as a therapeutic strategy against cancers harboring active PI3-K and Akt signaling pathways.

RNAi screening of the kinome with cytarabine in leukemias.

To identify rational therapeutic combinations with cytarabine (Ara-C), we developed a high-throughput, small-interference RNA (siRNA) platform for myeloid leukemia cells. Of 572 kinases individually silenced in combination with Ara-C, silencing of 10 (1.7%) and 8 (1.4%) kinases strongly increased Ara-C activity in TF-1 and THP-1 cells, respectively. The strongest molecular concepts emerged around kinases involved in cell-cycle checkpoints and DNA-damage repair. In confirmatory siRNA assays, inhibition of WEE1 resulted in more potent and universal sensitization across myeloid cell lines than siRNA inhibition of PKMYT1, CHEK1, or ATR. Treatment of 8 acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) cell lines with commercial and the first-in-class clinical WEE1 kinase inhibitor MK1775 confirmed sensitization to Ara-C up to 97-fold. Ex vivo, adding MK1775 substantially reduced viability in 13 of 14 AML, CML, and myelodysplastic syndrome patient samples compared with Ara-C alone. Maximum sensitization occurred at lower to moderate concentrations of both drugs. Induction of apoptosis was increased using a combination of Ara-C and MK1775 compared with using either drug alone. WEE1 is expressed in primary AML, ALL, and CML specimens. Data from this first siRNA-kinome sensitizer screen suggests that inhibiting WEE1 in combination with Ara-C is a rational combination for the treatment of myeloid and lymphoid leukemias.

High-throughput RNAi screening identifies a role for TNK1 in growth and survival of pancreatic cancer cells.

To identify novel targets in pancreatic cancer cells, we used high-throughput RNAi (HT-RNAi) to select genes that, when silenced, would decrease viability of pancreatic cancer cells. The HT-RNAi screen involved reverse transfecting the pancreatic cancer cell line BxPC3 with a siRNA library targeting 572 kinases. From replicate screens, approximately 32 kinases were designated as hits, of which 22 kinase targets were selected for confirmation and validation. One kinase identified as a hit from this screen was tyrosine kinase nonreceptor 1 (TNK1), a kinase previously identified as having tumor suppressor-like properties in embryonic stem cells. Silencing of TNK1 with siRNA showed reduced proliferation in a panel of pancreatic cancer cell lines. Furthermore, we showed that silencing of TNK1 led to increased apoptosis through a caspase-dependent pathway and that targeting TNK1 with siRNA can synergize with gemcitabine treatment. Despite previous reports that TNK1 affects Ras and NF-κB signaling, we did not find similar correlations with these pathways in pancreatic cancer cells. Our results suggest that TNK1 in pancreatic cancer cells does not possess the same tumor suppressor properties seen in embryonic cells but seems to be involved in growth and survival. The application of functional genomics by using HT-RNAi screens has allowed us to identify TNK1 as a growth-associated kinase in pancreatic cancer cells.

NCI60 cancer cell line panel data and RNAi analysis help identify EAF2 as a modulator of simvastatin and lovastatin response in HCT-116 cells.

Simvastatin and lovastatin are statins traditionally used for lowering serum cholesterol levels. However, there exists evidence indicating their potential chemotherapeutic characteristics in cancer. In this study, we used bioinformatic analysis of publicly available data in order to systematically identify the genes involved in resistance to cytotoxic effects of these two drugs in the NCI60 cell line panel. We used the pharmacological data available for all the NCI60 cell lines to classify simvastatin or lovastatin resistant and sensitive cell lines, respectively. Next, we performed whole-genome single marker case-control association tests for the lovastatin and simvastatin resistant and sensitive cells using their publicly available Affymetrix 125K SNP genomic data. The results were then evaluated using RNAi methodology. After correction of the p-values for multiple testing using False Discovery Rate, our results identified three genes (NRP1, COL13A1, MRPS31) and six genes (EAF2, ANK2, AKAP7, STEAP2, LPIN2, PARVB) associated with resistance to simvastatin and lovastatin, respectively. Functional validation using RNAi confirmed that silencing of EAF2 expression modulated the response of HCT-116 colon cancer cells to both statins. In summary, we have successfully utilized the publicly available data on the NCI60 cell lines to perform whole-genome association studies for simvastatin and lovastatin. Our results indicated genes involved in the cellular response to these statins and siRNA studies confirmed the role of the EAF2 in response to these drugs in HCT-116 colon cancer cells.

Amplification and overexpression of vinculin are associated with increased tumour cell proliferation and progression in advanced prostate cancer.

Androgen withdrawal is the standard treatment for advanced prostate cancer. Although this therapy is initially effective, nearly all prostate cancers become refractory to it. Approximately 15% of these castration-resistant prostate cancers harbour a genomic amplification at 10q22. The aim of this study was to explore the structure of the 10q22 amplicon and to determine the major driving genes. Application of high-resolution array-CGH using the 244k Agilent microarrays to cell lines with 10q22 amplification allowed us to narrow down the common amplified region to a region of 5.8 megabases. We silenced each of the genes of this region by an RNAi screen in the prostate cancer cell lines PC-3 and 22Rv1. We selected genes with a significant growth reduction in the 10q22 amplified cell line PC-3, but not in the non-amplified 22Rv1 cells, as putative target genes of this amplicon. Immunohistochemical analysis of the protein expression of these candidate genes on a tissue microarray enriched for 10q22 amplified prostate cancers revealed vinculin as the most promising target of this amplicon. We found a strong association between vinculin gene amplification and overexpression (p < 0.001). Further analysis of 443 specimens from across all stages of prostate cancer progression showed that vinculin expression was highest in castration-resistant prostate cancers, but negative or very low in benign prostatic hyperplasia (p < 0.0001). Additionally, high tumour cell proliferation measured by Ki67 expression was significantly associated with high vinculin expression in prostate cancer (p < 0.0001). Our data suggest that vinculin is a major driving gene of the 10q22 amplification in prostate cancer and that vinculin overexpression might contribute to prostate cancer progression by enhancing tumour cell proliferation.

Detailed molecular fingerprinting of four new anaplastic thyroid carcinoma cell lines and their use for verification of RhoB as a molecular therapeutic target.

CONTEXT: Anaplastic thyroid carcinoma (ATC) is a highly aggressive carcinoma in need of therapeutic options. One critical component of drug discovery is the availability of well-characterized cell lines for identification of molecular mechanisms related to tumor biology and drug responsiveness. Up to 42% of human thyroid cancer cell lines are redundant or not of correct tissue origin, and a comprehensive analysis is currently nonexistent. Mechanistically, RhoB has been identified as a novel molecular target for ATC therapy. OBJECTIVE: The aim was to develop four ATC cell lines detailing genetic, molecular, and phenotypic characteristics and to test five classes of drugs on the cell lines to determine whether they inhibited cell proliferation in a RhoB-dependent fashion. DESIGN: Four cell lines were derived from ATC tumors. Short tandem DNA repeat and mutational status of the originating tumors and cell lines were performed along with molecular and phenotypic characterizations. Compounds were tested for growth inhibition and ability to up-regulate RhoB. RESULTS: Cell line authenticity was confirmed by DNA short tandem repeat analysis. Each proved unique regarding expression of thyroid markers, oncogene status, amplified and deleted genes, and proliferative growth rates. FTI-277, GGTI-286, lovastatin, romidepsin, and UCN-01 up-regulated RhoB and inhibited cell proliferation in a dose-responsive fashion with only romidepsin and FTI-277 being RhoB dependent. CONCLUSIONS: Molecular descriptions of thyroid lines were matched to the originating tumors, setting a new standard for cell line characterization. Furthermore, suppressed RhoB is implicated as a molecular target for therapy against ATC because five classes of drugs up-regulate RhoB and inhibit growth dose-responsively.

RNAi screening of the kinome identifies modulators of cisplatin response in ovarian cancer cells.

OBJECTIVE: Ovarian cancer retains a poor prognosis among the female gynaecological malignancies. It constitutes about 3% of all malignancies in women and accounts for 5% of all female cancer related deaths. A standard treatment is cytoreductive surgery followed by adjuvant chemotherapy, and re-treatment with platinum based chemotherapy at the time of relapse. In order to improve cisplatin response in ovarian cancer cells, we utilized a high-throughput RNAi screening to identify kinase modulators. METHODS: A high-throughput RNAi screen was performed using a siRNA library targeting 572 kinases to identify potentiators of cisplatin response in the ovarian cancer cell line SKOV3. RESULTS: RNAi screening identified at least 55 siRNAs that potentiated the growth inhibitory effects of cisplatin in SKOV3 cells. Inhibition of ATR and CHK1 resulted in the greatest modulation of cisplatin response. Drug dose response of cisplatin in the presence of siRNA validated the effects of these target genes. To show that the siRNA data could be successfully translated into potential therapeutic strategies, CHK1 was further targeted with small molecule inhibitor PD 407824 in combination with cisplatin. Results showed that treatment of SKOV3 and OVCAR3 cells with CHK1 inhibitor PD 407824 led to sensitization of ovarian cancer cells to cisplatin. CONCLUSIONS: Our data provides kinase targets that could be exploited to design better therapeutics for ovarian cancer patients. We also demonstrate the effectiveness of high-throughput RNAi screening as a tool for identifying sensitizing targets to known and established chemotherapeutic agents.

RNAi phenotype profiling of kinases identifies potential therapeutic targets in Ewing's sarcoma.

BACKGROUND: Ewing's sarcomas are aggressive musculoskeletal tumors occurring most frequently in the long and flat bones as a solitary lesion mostly during the teen-age years of life. With current treatments, significant number of patients relapse and survival is poor for those with metastatic disease. As part of novel target discovery in Ewing's sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewing's sarcoma cells. RESULTS: Four Ewing's sarcoma cell lines TC-32, TC-71, SK-ES-1 and RD-ES were tested in high throughput-RNAi screens using a siRNA library targeting 572 kinases. Knockdown of 25 siRNAs reduced the growth of all four Ewing's sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewing's sarcoma cells as compared to normal fibroblasts. Secondary validation and preliminary mechanistic studies highlighted the kinases STK10 and TNK2 as having important roles in growth and survival of Ewing's sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis. CONCLUSION: In summary, RNAi-based phenotypic profiling proved to be a powerful gene target discovery strategy, leading to successful identification and validation of STK10 and TNK2 as two novel potential therapeutic targets for Ewing's sarcoma.

Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer.

The PAX3-FKHR fusion protein is present in a majority of alveolar rhabdomyosarcomas associated with increased aggressiveness and poor prognosis. To better understand the molecular pathogenesis of PAX3-FKHR, we carried out the first, unbiased genome-wide identification of PAX3-FKHR binding sites and associated target genes in alveolar rhabdomyosarcoma. The data shows that PAX3-FKHR binds to the same sites as PAX3 at both MYF5 and MYOD enhancers. The genome-wide analysis reveals that the PAX3-FKHR sites are (a) mostly distal to transcription start sites, (b) conserved, (c) enriched for PAX3 motifs, and (d) strongly associated with genes overexpressed in PAX3-FKHR-positive rhabdomyosarcoma cells and tumors. There is little evidence in our data set for PAX3-FKHR binding at the promoter sequences. The genome-wide analysis further illustrates a strong association between PAX3 and E-box motifs in these binding sites, suggestive of a common coregulation for many target genes. We also provide the first direct evidence that FGFR4 and IGF1R are the targets for PAX3-FKHR. The map of PAX3-FKHR binding sites provides a framework for understanding the pathogenic roles of PAX3-FKHR, as well as its molecular targets to allow a systematic evaluation of agents against this aggressive rhabdomyosarcoma.