Combinations of PARP Inhibitors with Temozolomide Drive PARP1 Trapping and Apoptosis in Ewing's Sarcoma.

Ewing's sarcoma is a malignant pediatric bone tumor with a poor prognosis for patients with metastatic or recurrent disease. Ewing's sarcoma cells are acutely hypersensitive to poly (ADP-ribose) polymerase (PARP) inhibition and this is being evaluated in clinical trials, although the mechanism of hypersensitivity has not been directly addressed. PARP inhibitors have efficacy in tumors with BRCA1/2 mutations, which confer deficiency in DNA double-strand break (DSB) repair by homologous recombination (HR). This drives dependence on PARP1/2 due to their function in DNA single-strand break (SSB) repair. PARP inhibitors are also cytotoxic through inhibiting PARP1/2 auto-PARylation, blocking PARP1/2 release from substrate DNA. Here, we show that PARP inhibitor sensitivity in Ewing's sarcoma cells is not through an apparent defect in DNA repair by HR, but through hypersensitivity to trapped PARP1-DNA complexes. This drives accumulation of DNA damage during replication, ultimately leading to apoptosis. We also show that the activity of PARP inhibitors is potentiated by temozolomide in Ewing's sarcoma cells and is associated with enhanced trapping of PARP1-DNA complexes. Furthermore, through mining of large-scale drug sensitivity datasets, we identify a subset of glioma, neuroblastoma and melanoma cell lines as hypersensitive to the combination of temozolomide and PARP inhibition, potentially identifying new avenues for therapeutic intervention. These data provide insights into the anti-cancer activity of PARP inhibitors with implications for the design of treatment for Ewing's sarcoma patients with PARP inhibitors.Research in the M.J.G. laboratory is supported by grants from the Wellcome Trust (086357 and 102696/Z/13/Z; http://www.wellcome.ac.uk/Funding). Research in the S.P.J. laboratory is funded by Cancer Research UK Program Grant C6/A11224 (http://www.cancerresearchuk.org/funding-for-researchers/our-funding-schemes), the European Research Council (http://erc.europa.eu/funding-and-grants)and the European Community Seventh Framework Program grant agreement no. HEALTH-F2-2010-259893 (DDResponse). Core infrastructure funding was provided by Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096. S.P.J. receives a salary from the University of Cambridge, supplemented by Cancer Research UK. J.T. was funded by the European Community Seventh Framework Program grant agreement no. HEALTH-F2-2010-259893 (DDResponse). U.M. is supported by a Cancer Research UK Clinician Scientist Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pone.014098

A suspension technique for efficient large-scale cancer organoid culturing and perturbation screens.

Organoid cell culture methodologies are enabling the generation of cell models from healthy and diseased tissue. Patient-derived cancer organoids that recapitulate the genetic and histopathological diversity of patient tumours are being systematically generated, providing an opportunity to investigate new cancer biology and therapeutic approaches. The use of organoid cultures for many applications, including genetic and chemical perturbation screens, is limited due to the technical demands and cost associated with their handling and propagation. Here we report and benchmark a suspension culture technique for cancer organoids which allows for the expansion of models to tens of millions of cells with increased efficiency in comparison to standard organoid culturing protocols. Using whole-genome DNA and RNA sequencing analyses, as well as medium-throughput drug sensitivity testing and genome-wide CRISPR-Cas9 screening, we demonstrate that cancer organoids grown as a suspension culture are genetically and phenotypically similar to their counterparts grown in standard conditions. This culture technique simplifies organoid cell culture and extends the range of organoid applications, including for routine use in large-scale perturbation screens

EWSCs are sensitive to PARP1 trapping.

<p><b>(A)</b> Relative viability of mock-transfected and PARP1 siRNA-transfected ES8 cells treated with vehicle or olaparib. Asterisks indicate <i>student’s paired t-test P</i> value **P<0.01, ns = not significant. <b>(B)</b> PARP1 expression in cells transfected with a scrambled control or a titration of PARP1_1 siRNA and their relative viability following treatment with vehicle or olaparib. <b>(C)</b> IC50 values of parental ES8 and PARPi-resistant OLAR5 cells to five different PARPi and the fold difference between them. <b>(D)</b> Western blot of PARP1 expression in ES8 and OLAR5 cells. Viability values are the mean of technical triplicates and representative of 3 independent experiments.</p

Temozolomide enhances PARP inhibitor sensitivity in multiple tumour types.

<p>List of cell lines screened against a combination of olaparib and temozolomide. Whether enhancement of PARP inhibitor sensitivity with temozolomide is observed (✔) or not (✖) is indicated.</p

DNA DSB repair by HR is functional in EWSCs.

<p><b>(A)</b> Western blot of ES8 cells treated with olaparib for the times indicated. Markers are grouped as part of ATM or ATR signaling. Tubulin served as a loading control. (<b>B)</b> Western blot of ES8 cells treated with camptothecin and harvested at various time points following drug washout. GAPDH served as a loading control. <b>(C)</b> Percentage of EdU-positive and EdU-negative ES8 cells with >5 nuclear RAD51 foci following 6-hour treatment with vehicle or olaparib (ola). <b>(D)</b> Olaparib log GI₅₀ (μM) of cell lines mock-transfected or transfected with CtIP or BRCA1 siRNA as indicated.</p

EWSCs are sensitive to PARP inhibition and S-phase DNA-damaging agents.

<p><b>(A)</b> and <b>(C)</b> Scatter plots of IC₅₀ (μM) values on a log scale comparing drug sensitivity of <i>EWS-FLI1</i>-positive and wild-type (WT) <i>EWS-FLI1</i>-negative cell lines to (A) four PARPi and (C) camptothecin and cisplatin. The sample size (n) is indicated and each circle represents the IC₅₀ of one cell line. The red bar is the geometric mean and the drug name is depicted above each plot along with the significance of the association as determined by an unpaired two-sample t-test. <b>(B)</b> Long term viability assays in EWSCs were performed in the presence of vehicle (-) or increasing concentrations of four PARPi as indicated. Non-EWSC lines (U-2-OS and HeLaSF) are included for comparison. These data are representative of 3 independent experiments.</p

Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists

Multivalent second-generation TRAIL-R2 agonists are currently in late pre-clinical development and early clinical trials. Herein, we use a representative 2(nd) generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models. Whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to supress MEDI3039 induced cell-death. High throughput drug-screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics anaysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell-lines and patient-derived xenografts, identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination-screening of TRAIL-R2, caspase-8, FADD and BID knockout models with 60 compounds with varying mechanisms-of-action identified 2 inhibitor of apoptosis proteins (IAPs) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8: MCL-1 as potential predictive biomarkers for second generation TRAIL-R2 agonists and loss of key effectors like FADD and caspase-8 as likely drivers of clinical resistance in solid tumors

Genome-wide chemical mutagenesis screens allow unbiased saturation of the cancer genome and identification of drug resistance mutations

Drug resistance is an almost inevitable consequence of cancer therapy and ultimately proves fatal for the majority of patients. In many cases, this is the consequence of specific gene mutations that have the potential to be targeted to resensitize the tumor. The ability to uniformly saturate the genome with point mutations without chromosome or nucleotide sequence context bias would open the door to identify all putative drug resistance mutations in cancer models. Here, we describe such a method for elucidating drug resistance mechanisms using genome-wide chemical mutagenesis allied to next-generation sequencing. We show that chemically mutagenizing the genome of cancer cells dramatically increases the number of drug-resistant clones and allows the detection of both known and novel drug resistance mutations. We used an efficient computational process that allows for the rapid identification of involved pathways and druggable targets. Such a priori knowledge would greatly empower serial monitoring strategies for drug resistance in the clinic as well as the development of trials for drug-resistant patients

Effective drug combinations in breast, colon and pancreatic cancer cells.

Combinations of anti-cancer drugs can overcome resistance and provide new treatments1,2. The number of possible drug combinations vastly exceeds what could be tested clinically. Efforts to systematically identify active combinations and the tissues and molecular contexts in which they are most effective could accelerate the development of combination treatments. Here we evaluate the potency and efficacy of 2,025 clinically relevant two-drug combinations, generating a dataset encompassing 125 molecularly characterized breast, colorectal and pancreatic cancer cell lines. We show that synergy between drugs is rare and highly context-dependent, and that combinations of targeted agents are most likely to be synergistic. We incorporate multi-omic molecular features to identify combination biomarkers and specify synergistic drug combinations and their active contexts, including in basal-like breast cancer, and microsatellite-stable or KRAS-mutant colon cancer. Our results show that irinotecan and CHEK1 inhibition have synergistic effects in microsatellite-stable or KRAS-TP53 double-mutant colon cancer cells, leading to apoptosis and suppression of tumour xenograft growth. This study identifies clinically relevant effective drug combinations in distinct molecular subpopulations and is a resource to guide rational efforts to develop combinatorial drug treatments