Irma M. Gonzales

Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

BackgroundPancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement.MethodsIn order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine.ResultsResults from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1). Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine.ConclusionThese findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer cells to gemcitabine.

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.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

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. Copyright

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. Mol Cancer Res; 9(6); 724–32. ©2011 AACR.

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

BackgroundEwings 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 Ewings sarcoma, we applied RNAi mediated phenotypic profiling to identify kinase targets involved in growth and survival of Ewings sarcoma cells.ResultsFour Ewings 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 Ewings sarcoma cell lines in replicate screens. Of these, 16 siRNA were specific and reduced proliferation of Ewings 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 Ewings sarcoma cells. Furthermore, knockdown of STK10 and TNK2 by siRNA showed increased apoptosis.ConclusionIn 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 Ewings sarcoma.

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

Summary 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.

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.

Chemogenomic analysis identifies Macbecin II as a compound specific for SMAD4-negative colon cancer cells.

The tumor suppressor gene, SMAD4, is mutated in approximately 30% of colon cancers. To identify compounds with enhanced potency on cells with a SMAD4‐negative context, we combined genomic and cheminformatic analyses of publicly available data relating to the colon cancer cell lines within the NCI60 panel. Two groups of cell lines were identified with either wild‐type or negative SMAD4 status. A cheminformatic analysis of the NCI60 screening data was carried out, which led to the identification of 14 compounds that preferentially inhibited cell growth of the SMAD4‐negative cell lines. Using cell viability assays, the effect of these compounds was validated on four colon cancer cell lines: HCT‐116 and HCT‐15 (SMAD4‐expressing), and HT‐29 and COLO‐205 (SMAD4‐negative). Our data identified Macbecin II, a hydroquinone ansamycin antibiotic, as having increased potency in the SMAD4‐negative cells compared to SMAD4 wild‐type cells. In addition, we showed that silencing of SMAD4 using siRNA in HCT‐116 enhanced Macbecin II potency. Our results demonstrate that Macbecin II is specifically active in colon cancer cells having a SMAD4‐negative background and thus is a potential candidate for further investigation in a drug discovery perspective.

Abstract LB-128: Synthetic lethal RNAi screening identifies inhibition of Bcl-2 family members as sensitizers to 5-Azacytidine in myeloid cells

In order to identify novel targets and clinically exploitable vulnerabilities, we performed high-throughput RNA-interference (RNAi) synthetic lethality screening using myeloid cell lines TF-1 and ML-2 in combination with the hypomethylating agent 5-Azacytidine. A custom small interfering RNA (siRNA) library targeting nearly 1000 cancer-associated genes, including the human kinome, was utilized. Bcl-XL emerged as the strongest 5-Azacytidine sensitizing target in the erythroid cell line TF-1 but not in ML-2 cells. Additional siRNA sequences targeting Bcl-XL were transfected into a panel of myeloid cell lines and tested in drug dose response experiments using a ten-point serial dilution of 5-Azacytidine. The erythroid differentiated myeloid cell lines TF-1 and HEL both exhibited a 3 to 4-fold reduction in the IC50 of 5-Azacytidine upon knockdown of Bcl-XL, whereas IC50 values for non-erythroid, myeloid cell lines ML-2, THP-1 and MDS-L were unchanged. Furthermore, we tested lentiviral delivery of short-hairpin RNA (shRNA) targeting Bcl-XL in additional cell lines. Lentiviral knockdown of Bcl-XL confirmed 2 to 3-fold 5-Azacytidine sensitization in TF-1 and HEL cells while U937, HL-60, ML-2, THP-1 and MDS-L were not sensitized by Bcl-XL knockdown. Interestingly, while Bcl-XL knockdown with shRNA potentiated the effects of 5-Azacytidine in TF-1 and HEL, Bcl-XL knockdown alone resulted in considerable reductions in cell viability in TF-1 and HEL cells. These results demonstrate erythroid lineage-specific responses to Bcl-XL knockdown and suggest that Bcl-XL may represent an erythroid lineage-specific therapeutic vulnerability. To begin to explore this possibility we examined the putative BH3 mimetics Gossypol, Obatoclax and ABT-737 with 5-Azacytidine drug dose response experiments in myeloid cell lines. Neither Gossypol nor Obatoclax potentiated the effects of 5-Azacytdine. In striking contrast, ABT-737 resulted in a 2 to 4-fold reduction in the IC50 of 5-Azacytidine in all cell lines tested to date (TF-1, HEL, ML-2 and THP-1). ABT-737 sensitization to 5-Azacytidine was dose dependent. Furthermore, Combination Index (CI) values calculated with Calcusyn software demonstrate synergy between ABT-737 and 5-Azacytidine at clinically relevant concentrations. These results suggest that inhibition of anti-apoptotic Bcl-2 family members with a BH3 mimetic may represent an effective strategy for 5-Azacytidine combination therapy that can be directly translated into design of combination clinical trials in patients with myeloid leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-128.