High-throughput identification of genotype-specific cancer vulnerabilities in mixtures of barcoded tumor cell lines.

Hundreds of genetically characterized cell lines are available for the discovery of genotype-specific cancer vulnerabilities. However, screening large numbers of compounds against large numbers of cell lines is currently impractical, and such experiments are often difficult to control. Here we report a method called PRISM that allows pooled screening of mixtures of cancer cell lines by labeling each cell line with 24-nucleotide barcodes. PRISM revealed the expected patterns of cell killing seen in conventional (unpooled) assays. In a screen of 102 cell lines across 8,400 compounds, PRISM led to the identification of BRD-7880 as a potent and highly specific inhibitor of aurora kinases B and C. Cell line pools also efficiently formed tumors as xenografts, and PRISM recapitulated the expected pattern of erlotinib sensitivity in vivo

The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.

Encapsulation of drugs within nanocarriers that selectively target malignant cells promises to mitigate side effects of conventional chemotherapy and to enable delivery of the unique drug combinations needed for personalized medicine. To realize this potential, however, targeted nanocarriers must simultaneously overcome multiple challenges, including specificity, stability and a high capacity for disparate cargos. Here we report porous nanoparticle-supported lipid bilayers (protocells) that synergistically combine properties of liposomes and nanoporous particles. Protocells modified with a targeting peptide that binds to human hepatocellular carcinoma exhibit a 10,000-fold greater affinity for human hepatocellular carcinoma than for hepatocytes, endothelial cells or immune cells. Furthermore, protocells can be loaded with combinations of therapeutic (drugs, small interfering RNA and toxins) and diagnostic (quantum dots) agents and modified to promote endosomal escape and nuclear accumulation of selected cargos. The enormous capacity of the high-surface-area nanoporous core combined with the enhanced targeting efficacy enabled by the fluid supported lipid bilayer enable a single protocell loaded with a drug cocktail to kill a drug-resistant human hepatocellular carcinoma cell, representing a 10(6)-fold improvement over comparable liposomes

Evaluation of the complexity of cell cycle effects induced by anticancer drugs

The time- and dose-dependence of the blocking activity, cell loss and recycling from block that follow a treatment with anticancer agents is not always easy to interpret, especially because of the superimposition of cytostatic and cytotoxic effects. Their separate quantification, in terms of the cellular control mechanisms that regulate the response to a drug treatment, is important to deeply understand the mode of action of an anticancer compound. We investigated cell cycle effects induced by different drugs on a cell line growing in vitro with an approach including experimental data coming from cell counting and different flow cytometric techniques and a mathematical model reproducing the perturbed cell growth after drug treatment. In this way we could quantify the effects induced by treatments performed with topotecan, doxorubicin and melphalan on IGROV1 cells, evaluating separately cytostatic and cytotoxic effects. For each one of the considered drugs we obtained and compared a set of parameters describing the time- and dose-dependence of cell cycle effects. These results were compared with those previously published by our group (Sena et al., 1999; Montalenti et al., 1998). Besides the classical flow cytometric techniques, we used the method of carboxyfluorescein diacetate succinimidyl ester staining to quantify and appreciate the heterogeneity of the effects induced by a treatment on the cells of the same population. The results obtained could be simulated with our mathematical model. In the last part of the project, the previously listed tools were used to obtain two sets of parameters describing the effects induced by a short treatment of cisplatin on HCT-116 and HCT-116 p53-/-. This allowed the quantification of the role of p53 in the cell response to cisplatin treatment, finding that this protein has a more important role in the activation of the apoptosis than in the cytostatic effects induced by this drug

Experimental determination of the optimal means of combining topotecan and (131 I) MIBG therapies for the treatment of neuroblastoma

Introduction: Neuroblastoma has a long-term survival rate of only 15%. While patients with early stage disease can usually be treated surgically, those with inoperable disease require intensive treatment. However, there has been no substantial improvement in the survival rates of patients with advanced disease. Targeted radiotherapy, using [131I] meta-iodobenzylguanidine ([131I]MIBG) has induced favourable remissions in some patients when used as a single agent. However, the full potential of this therapy may only be realised when it is combined with other agents. One class of agents with the potential to improve [131I]MIBG therapy are the inhibitors of topoisomerase I, which have previously shown synergy when used in combination with radiotherapy. Furthermore [131I]MIBG therapy could be improved by the rapid selection of patients who may derive benefit from this treatment. Aims: The aims of this study were to develop a molecular assay for MIBG active uptake, and to determine the efficacy of [131I]MIBG in combination with the topoisomerase I inhibitor topotecan in vitro and in vivo. Results: RT-PCR analysis of NAT expression by neuroblastoma biopsies was predictive of tumour uptake of MIBG. However RT-PCR negativity failed to correctly determine MIBG uptake capacity with a frequency of 45%. The combined effects of topotecan and [131I]MIBG were assessed, in tumour cells expressing the noradrenaline transporter, using three treatment schedules: topotecan administered 24h before [i], after [ii] or simultaneously with [iii] [131I]MIBG. Pre-treatment with TPT enhanced the intracellular concentration of [131I]MIBG. However, analysis of TPT and [131I]MIBG interactions in vitro and in vivo demonstrated that this was not the optimal order of administration for combination therapy. While supra-additive toxicity was observed in vitro by all combination schedules, schedule [i] was less effective than schedules [ii] and [iii]. This was reflected in failure to repair DNA damage. With respect to delay of growth of NAT-expressing xenografts in nude mice, combinations of topotecan and [131I]MIBG were more effective than single agent treatments. Combination schedules [ii] and [iii] were superior to combination schedule [i]. Combination treatment caused negligible myelotoxicity according to platelet production, or stem cell clonogenic capacity. Conclusions: Real-time PCR evaluation of primary neuroblastoma tumours has significant capability to reflect their capacity for the accumulation of MIBG, however PCR-based assessment of NAT gene expression cannot fully predict MIBG uptake. Inhibition of DNA repair and supra-additive toxicity to NAT expressing cells and xenografts were achieved using combinations of topotecan and [131I]MIBG. Effectiveness was dependent on the order of administration of the two agents. If the synergy demonstrated in the model systems used in this study can be replicated in patients with neuroblastoma, there is potential for real therapeutic gain

Strategies for surviving down-regulation : effects on tumour cell growth potential and chemosensitivity profile

Survivin is a bifunctional protein that acts as a suppressor of apoptosis and plays a central role in cell division. The protein is strongly expressed in the most common human neoplasms, has prognostic relevance for some of them, and appears to be involved in tumour cell resistance to anticancer agents and ionizing radiation. On the basis of these findings, survivin has been proposed as an attractive target for new anticancer interventions. We generated a hammerhead ribozyme (Rz) targeting the CUA110 triplet in the survivin mRNA and transfected them into the JR8 human melanoma cell line. Cells endogenously expressing Rz were characterized by a lower survivin protein level than parental cells, and showed an increased caspase-9-dependent apoptotic response to treatment with the cytotoxic agents cisplatin and topotecan as well as with ɣ-irradiation. Moreover, an increased anti-tumour activity of oral topotecan was observed in Rz-expressing cells grown as xenograft tumours in athymic nude mice. In addition, we constructed a Moloney-based retroviral vector expressing Rz, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations, obtained by infection with the retroviral vector, of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were characterized by a significant reduction of survivin expression; the cells became polyploid and underwent caspase-9-dependent apoptosis. Survivin inhibition also enhanced their susceptibility to cisplatin-induced apoptosis and prevented tumour formation when cells were xenografted into athymic nude mice. Again we used RNAi to specifically repress survivin in DU145 and PC-3 cell lines. RNAi-mediated survivin knock-down was able to significantly reduce cell proliferation and to enhance the rate of caspase-9-dependent apoptosis. Moreover, sequential treatment with survivin-specific siRNA followed by the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin produced supra-additive anti-proliferative effects in both cell lines. Finally, we investigated the effects of the novel cdk inhibitor NU6140, in term of ability to potentiate the response to paclitaxel in HeLa cells, in relation to its interference with survivin. Sequential administration of cdk inhibitors resulted in escape from the mitotic block imposed by paclitaxel and significantly increased the apoptotic rate, with inhibition of survivin expression/phosphorylation as the potential mechanism. Overall, such results suggest that strategies aimed at interfering with survivin expression/activity can be adopted to improve the chemo/radio-sensitivity profile of treatment-refractory human malignancies

Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small cell lung cancer cells

Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5'-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations

Targeted Therapies for Ovarian Cancer

Epithelial ovarian cancer has the highest mortality rate of all gynaecological malignancies. Most women present with advanced disease and develop a recurrence after radical surgery and chemotherapy. Improving the results of first- or subsequent-line chemotherapy has been slow, and novel approaches to systemic treatment are needed. Ovarian cancer is a heterogeneous disease with complex molecular and genetic changes. Understanding these better will provide information on the mechanisms of resistance and opportunities to target therapy more rationally, exploiting specific changes in the tumour. Here we reviewed targeted approaches to therapy, focussing on targeting angiogenesis and inhibition of DNA repair, 2 areas that show promising activity. Additionally, we reviewed studies that are underway, targeting the cell cycle, signalling pathways and immunotherapeutic strategies. Many of these innovative approaches already demonstrate promising activity in ovarian cancer and have the potential to improve the outcome in women with ovarian cancer

Rationalising Multi-Agent Chemotherapy through Systems Analysis of Drug-Induced Apoptosis for High-Risk Neuroblastoma

High-risk neuroblastoma is an aggressive and invasive paediatric malignancy, with few actionable somatic mutations. As such attempts to implement precision medicine based approaches have been limited and intense multi-agent chemotherapy remains the standard-of-care. This project applied a systems biology approach to perform integrated proteomic and functional analysis of chemotherapy induced apoptosis to identify and define the emergence of synergy between relevant chemotherapy drug combinations. Firstly, a functional genomics screen was performed on a high content cellomics platform with a siRNA library of 200 apoptotic genes with current standard-of-care chemotherapy and preclinical drugs. Multi-dimensional analysis of this dataset elegantly demonstrated that synergy between any two chemotherapy drugs is proportional to the magnitude of divergence in apoptotic signalling between individual drugs. In particular, romidepsin was identified as an apoptotically distinct chemotherapy with universal drug synergy within the investigative drug panel. Identified key drug-specific apoptotic signalling nodes underlying this synergy were validated in vitro using genetically incorporated fluorescent biosensors, endogenously tagging and multiplexed IF. These tools allowed us to perform high-throughput kinetic live cell analysis at the single cell resolution. Optimised drug combinations were further validated in vivo using matched PDXs of treatment naïve and relapsed tumours. Collectively, this project has demonstrated that synergistic combinations emerge from the differential utilization of apoptotic signaling pathways by each single agent, regardless of the direct molecular target of each drug. This is contrary to the current dogma of utilising drugs with different molecular targets in combination chemotherapy. The results of this work contribute to developing non-traditional precision medicine approaches to rationalise multi-agent chemotherapy by characterising single agent apoptotic signalling

Interactions of human multidrug resistance protein 4 with camptothecins

Ph.DDOCTOR OF PHILOSOPH

Inhibition of Post-Transcriptional Steps in Ribosome Biogenesis Confers Cytoprotection Against Chemotherapeutic Agents in a p53-Dependent Manner

The p53-mediated nucleolar stress response associated with inhibition of ribosomal RNA transcription was previously shown to potentiate killing of tumor cells. Here, we asked whether targeting of ribosome biogenesis can be used as the basis for selective p53-dependent cytoprotection of nonmalignant cells. Temporary functional inactivation of the 60S ribosome assembly factor Bop1 in a 3T3 cell model markedly increased cell recovery after exposure to camptothecin or methotrexate. This was due, at least in part, to reversible pausing of the cell cycle preventing S phase associated DNA damage. Similar cytoprotective effects were observed after transient shRNA-mediated silencing of Rps19, but not several other tested ribosomal proteins, indicating distinct cellular responses to the inhibition of different steps in ribosome biogenesis. By temporarily inactivating Bop1 function, we further demonstrate selective killing of p53-deficient cells with camptothecin while sparing isogenic p53-positive cells. Thus, combining cytotoxic treatments with inhibition of select post-transcriptional steps of ribosome biogenesis holds potential for therapeutic targeting of cells that have lost p53