Showing papers by "Yves Pommier published in 2015"

Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins

Abstract: The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained γ-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens.

Using CellMiner 1.6 for Systems Pharmacology and Genomic Analysis of the NCI-60

Abstract: The NCI-60 cancer cell line panel provides a premier model for data integration, and systems pharmacology being the largest publicly available database of anticancer drug activity, genomic, molecular, and phenotypic data. It comprises gene expression (25,722 transcripts), microRNAs (360 miRNAs), whole-genome DNA copy number (23,413 genes), whole-exome sequencing (variants for 16,568 genes), protein levels (94 genes), and cytotoxic activity (20,861 compounds). Included are 158 FDA-approved drugs and 79 that are in clinical trials. To improve data accessibility to bioinformaticists and non-bioinformaticists alike, we have developed the CellMiner web-based tools. Here, we describe the newest CellMiner version, including integration of novel databases and tools associated with whole-exome sequencing and protein expression, and review the tools. Included are (i) "Cell line signature" for DNA, RNA, protein, and drugs; (ii) "Cross correlations" for up to 150 input genes, microRNAs, and compounds in a single query; (iii) "Pattern comparison" to identify connections among drugs, gene expression, genomic variants, microRNA, and protein expressions; (iv) "Genetic variation versus drug visualization" to identify potential new drug:gene DNA variant relationships; and (v) "Genetic variant summation" designed to provide a synopsis of mutational burden on any pathway or gene group for up to 150 genes. Together, these tools allow users to flexibly query the NCI-60 data for potential relationships between genomic, molecular, and pharmacologic parameters in a manner specific to the user's area of expertise. Examples for both gain- (RAS) and loss-of-function (PTEN) alterations are provided.

SLFN11 Is a Transcriptional Target of EWS-FLI1 and a Determinant of Drug Response in Ewing Sarcoma

Abstract: Purpose: SLFN11 was identified as a critical determinant of response to DNA targeted therapies by analyzing gene expression and drug sensitivity of NCI-60 and CCLE datasets. However, how SLFN11 is regulated in cancer cells remained unknown. Ewing9s sarcoma (ES), which is characterized by the chimeric transcription factor EWS-FLI1, has notably high SLFN11 expression, leading us to investigate whether EWS-FLI1 drives SLFN11 expression and the role of SLFN11 in the drug response of ES cells. Experimental Design: Binding sites of EWS-FLI1 on the SLFN11 promoter were analyzed by chromatin immunoprecipitation-DNA sequence (ChIP-Seq) and promoter-luciferase reporter analyses. The relationship between SLFN11 and EWS-FLI1 were further examined in EWS-FLI1-knockdown or -overexpressing cells and in clinical tumor samples. Results: EWS-FLI1 binds near the transcription start site of SLFN11 promoter and acts as a positive regulator of SLFN11 expression in ES cells. EWS-FLI1-mediated SLFN11 expression is responsible for high sensitivity of ES to camptothecin and combinations of PARP inhibitors with temozolomide. Importantly, ES patients with higher SLFN11 expression showed better tumor-free survival rate. The correlated expression between SLFN11 and FLI1 extends to leukemia, pediatric, colon, breast and prostate cancers. In addition, expression of other ETS members correlates with SLFN11 in NCI-60 and CCLE datasets, and molecular experiments demonstrate that ETS1 acts as a positive regulator for SLFN11 expression in breast cancer cells. Conclusions: Our results imply the emerging relevance of SLFN11 as an ETS transcription factor response gene and for therapeutic response to topoisomerase I inhibitors and temozolomide-PARP inhibitor combinations in ETS-activated cancers.

Structure–Activity Relationship of Pyrrolyl Diketo Acid Derivatives as Dual Inhibitors of HIV-1 Integrase and Reverse Transcriptase Ribonuclease H Domain

Abstract: The development of HIV-1 dual inhibitors is a highly innovative approach aimed at reducing drug toxic side effects as well as therapeutic costs. HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) are both selective targets for HIV-1 chemotherapy, and the identification of dual IN/RNase H inhibitors is an attractive strategy for new drug development. We newly synthesized pyrrolyl derivatives that exhibited good potency against IN and a moderate inhibition of the RNase H function of RT, confirming the possibility of developing dual HIV-1 IN/RNase H inhibitors and obtaining new information for the further development of more effective dual HIV-1 inhibitors.

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

Abstract: The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81-deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81-deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins.

Lack of mitochondrial topoisomerase I (TOP1mt) impairs liver regeneration

Abstract: The liver has an exceptional replicative capacity following partial hepatectomy or chemical injuries Cellular proliferation requires increased production of energy and essential metabolites, which critically depend on the mitochondria To determine whether Top1mt, the vertebrate mitochondrial topoisomerase, is involved in this process, we studied liver regeneration after carbon tetrachloride (CCl4) administration TOP1mt knockout (KO) mice showed a marked reduction in regeneration and hepatocyte proliferation The hepatic mitochondrial DNA (mtDNA) failed to increase during recovery from CCl4 exposure Reduced glutathione was also depleted, indicating increased reactive oxygen species (ROS) Steady-state levels of ATP, O2 consumption, mtDNA, and mitochondrial mass were also reduced in primary hepatocytes from CCl4-treated KO mice To further test whether Top1mt acted by enabling mtDNA regeneration, we tested TOP1mt KO fibroblasts and human colon carcinoma HCT116 cells and measured mtDNA after 3-d treatment with ethidium bromide Both types of TOP1mt knockout cells showed defective mtDNA regeneration following mtDNA depletion Our study demonstrates that Top1mt is required for normal mtDNA homeostasis and for linking mtDNA expansion with hepatocyte proliferation

Synthesis and biological evaluation of nitrated 7-, 8-, 9-, and 10-hydroxyindenoisoquinolines as potential dual topoisomerase I (Top1)-tyrosyl-DNA phosphodiesterase I (TDP1) inhibitors.

Abstract: The structure–activity relationships and hit-to-lead optimization of dual Top1–TDP1 inhibitors in the indenoisoquinoline drug class were investigated. A series of nitrated 7-, 8-, 9-, and 10-hydroxyindenoisoquinolines were synthesized and evaluated. Several compounds displayed potent dual Top1–TDP1 inhibition. The 9-hydroxy series exhibited potencies and cytotoxicities vs Top1 that surpassed those of camptothecin (CPT), the natural alkaloid that is being used as a standard in the Top1-mediated DNA cleavage assay. One member of this series was a more potent Top1 inhibitor at a concentration of 5 nM and produced a more stable ternary drug–DNA–Top1 cleavage complex than CPT.

Relative contribution of four nucleases, CtIP, Dna2, Exo1 and Mre11, to the initial step of DNA double-strand break repair by homologous recombination in both the chicken DT40 and human TK6 cell lines.

Abstract: Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR.

The POLD3 subunit of DNA polymerase δ can promote translesion synthesis independently of DNA polymerase ζ

Abstract: The replicative DNA polymerase Polδ consists of a catalytic subunit POLD1/p125 and three regulatory subunits POLD2/p50, POLD3/p66 and POLD4/p12. The ortholog of POLD3 in Saccharomyces cerevisiae, Pol32, is required for a significant proportion of spontaneous and UV-induced mutagenesis through its additional role in translesion synthesis (TLS) as a subunit of DNA polymerase ζ. Remarkably, chicken DT40 B lymphocytes deficient in POLD3 are viable and able to replicate undamaged genomic DNA with normal kinetics. Like its counterpart in yeast, POLD3 is required for fully effective TLS, its loss resulting in hypersensitivity to a variety of DNA damaging agents, a diminished ability to maintain replication fork progression after UV irradiation and a significant decrease in abasic site-induced mutagenesis in the immunoglobulin loci. However, these defects appear to be largely independent of Polζ, suggesting that POLD3 makes a significant contribution to TLS independently of Polζ in DT40 cells. Indeed, combining polη, polζ and pold3 mutations results in synthetic lethality. Additionally, we show in vitro that POLD3 promotes extension beyond an abasic by the Polδ holoenzyme suggesting that while POLD3 is not required for normal replication, it may help Polδ to complete abasic site bypass independently of canonical TLS polymerases.

Discovery of potent indenoisoquinoline topoisomerase I poisons lacking the 3-nitro toxicophore.

Abstract: 3-Nitroindenoisoquinoline human topoisomerase IB (Top1) poisons have potent antiproliferative effects on cancer cells. The undesirable nitro toxicophore could hypothetically be replaced by other functional groups that would retain the desired biological activities and minimize potential safety risks. Eleven series of indenoisoquinolines bearing 3-nitro bioisosteres were synthesized. The molecules were evaluated in the Top1-mediated DNA cleavage assay and in the National Cancer Institute’s 60 cell line cytotoxicity assay. The data reveal that fluorine and chlorine may substitute for the 3-nitro group with minimal loss of Top1 poisoning activity. The new information gained from these efforts can be used to design novel indenoisoquinolines with improved safety.

N-Substituted Quinolinonyl Diketo Acid Derivatives as HIV Integrase Strand Transfer Inhibitors and Their Activity against RNase H Function of Reverse Transcriptase.

Abstract: Bifunctional quinolinonyl DKA derivatives were first described as nonselective inhibitors of 3′-processing (3′-P) and strand transfer (ST) functions of HIV-1 integrase (IN), while 7-aminosubstituted quinolinonyl derivatives were proven IN strand transfer inhibitors (INSTIs) that also displayed activity against ribonuclease H (RNase H). In this study, we describe the design, synthesis, and biological evaluation of new quinolinonyl diketo acid (DKA) derivatives characterized by variously substituted alkylating groups on the nitrogen atom of the quinolinone ring. Removal of the second DKA branch of bifunctional DKAs, and the amino group in position 7 of quinolinone ring combined with a fine-tuning of the substituents on the benzyl group in position 1 of the quinolinone, increased selectivity for IN ST activity. In vitro, the most potent compound was 11j (IC50 = 10 nM), while the most active compounds against HIV infected cells were ester derivatives 10j and 10l. In general, the activity against RNase H was n...

Using drug response data to identify molecular effectors, and molecular "omic" data to identify candidate drugs in cancer

Abstract: The current convergence of molecular and pharmacological data provides unprecedented opportunities to gain insights into the relationships between the two types of data. Multiple forms of large-scale molecular data, including but not limited to gene and microRNA transcript expression, DNA somatic and germline variations from next-generation DNA and RNA sequencing, and DNA copy number from array comparative genomic hybridization are all potentially informative when one attempts to recognize the panoply of potentially influential events both for cancer progression and therapeutic outcome. Concurrently, there has also been a substantial expansion of the pharmacological data being accrued in a systematic fashion. For cancer cell lines, the National Cancer Institute cell line panel (NCI-60), the Cancer Cell Line Encyclopedia (CCLE), and the collaborative Genomics of Drug Sensitivity in Cancer (GDSC) databases all provide subsets of these forms of data. For the patient-derived data, The Cancer Genome Atlas (TCGA) provides analogous forms of genomic information along with treatment histories. Integration of these data in turn relies on the fields of statistics and statistical learning. Multiple algorithmic approaches may be chosen, depending on the data being considered, and the nature of the question being asked. Combining these algorithms with prior biological knowledge, the results of molecular biological studies, and the consideration of genes as pathways or functional groups provides both the challenge and the potential of the field. The ultimate goal is to provide a paradigm shift in the way that drugs are selected to provide a more targeted and efficacious outcome for the patient.

Classification of PARP Inhibitors Based on PARP Trapping and Catalytic Inhibition, and Rationale for Combinations with Topoisomerase I Inhibitors and Alkylating Agents

Abstract: All PARP inhibitors in clinical development (veliparib, olaparib, niraparib, rucaparib, talazoparib) are potent submicromolar competitive NAD+ inhibitors for PARP1 and PARP2, thereby blocking PARylation reactions [i.e. formation of poly(ADPribose) polymers]. In addition, PARP trapping, which determines the anticancer activity of PARP inhibitors as single agents, is drug-specific, and PARP inhibitors can be ranked according to their PARP trapping potency: Talazoparib >> niraparib ˜ olaparib ˜ rucaparib > veliparib. The highly synergistic effects of PARP inhibitors in combination with alkylating agent (temozolomide or methyl methanesulfonate, MMS) and topoisomerase I (Top1) inhibitors (camptothecins and indenoisoquinolines) are well documented. Both classes of drugs induce DNA single-strand breaks sensed by PARP. Yet, the molecular mechanisms of synergy are different. For alkylating agents (temozolomide and MMS), both PARP trapping and PARylation inhibition account for the synergy, whereas for Top1 inhibitors, there is no involvement of PARP trapping and it is PARylation inhibition that deters the coupling of PARP with the repair enzyme, tyrosyl-DNA phosphodiesterase TDP1. In this chapter, we will review the differences between PARP inhibitors and the rationale for choosing among different PARP inhibitors in combination with alkylating agents or Top1 inhibitors.

Activation of RAF1 (c-RAF) by the Marine Alkaloid Lasonolide A Induces Rapid Premature Chromosome Condensation.

Abstract: Lasonolide A (LSA), a potent antitumor polyketide from the marine sponge, Forcepia sp., induces rapid and reversible protein hyperphosphorylation and premature chromosome condensation (PCC) at nanomolar concentrations independent of cyclin-dependent kinases. To identify cellular targets of LSA, we screened 2951 shRNAs targeting a pool of human kinases and phosphatases (1140 RefSeqs) to identify genes that modulate PCC in response to LSA. This led to the identification of RAF1 (C-RAF) as a mediator of LSA-induced PCC, as shRNAs against RAF1 conferred resistance to LSA. We found that LSA induced RAF1 phosphorylation on Serine 338 within minutes in human colorectal carcinoma HCT-116, ovarian carcinoma OVCAR-8, and Burkitt’s lymphoma CA46 cell lines. RAF1 depletion by siRNAs attenuated LSA-induced PCC in HCT-116 and OVCAR-8 cells. Furthermore, mouse embryonic fibroblasts (MEF) with homozygous deletion in Raf1, but not deletion in the related kinase Braf, were resistant to LSA-induced PCC. Complementation of Raf1−/− MEFs with wild-type human RAF1, but not with kinase-dead RAF1 mutant, restored LSA-induced PCC. Finally, the Raf inhibitor sorafenib, but not the MEK inhibitor AZD6244, effectively suppressed LSA-induced PCC. Our findings implicate a previously unknown, MAPK-independent role of RAF1 in chromatin condensation and potent activation of this pathway by LSA.

Inhibitory Effect of the Noncamptothecin Topoisomerase I Inhibitor LMP-400 on Female Mice Models and Human Pheochromocytoma Cells.

Abstract: Metastatic pheochromocytoma continues to be an incurable disease, and treatment with conventional cytotoxic chemotherapy offers limited efficacy. In the present study, we evaluated a novel topoisomerase I inhibitor, LMP-400, as a potential treatment for this devastating disease. We found a high expression of topoisomerase I in human metastatic pheochromocytoma, providing a basis for the evaluation of a topoisomerase 1 inhibitor as a therapeutic strategy. LMP-400 inhibited the cell growth of established mouse pheochromocytoma cell lines and primary human tumor tissue cultures. In a study performed in athymic female mice, LMP-400 demonstrated a significant inhibitory effect on tumor growth with two drug administration regimens. Furthermore, low doses of LMP-400 decreased the protein levels of hypoxia-inducible factor 1 (HIF-1α), one of a family of factors studied as potential metastatic drivers in these tumors. The HIF-1α decrease resulted in changes in the mRNA levels of HIF-1 transcriptional targets. In vitro, LMP-400 showed an increase in the growth-inhibitory effects in combination with other chemotherapeutic drugs that are currently used for the treatment of pheochromocytoma. We conclude that LMP-400 has promising antitumor activity in preclinical models of metastatic pheochromocytoma and its use should be considered in future clinical trials.

Single-Molecule Supercoil Relaxation Assay as a Screening Tool to Determine the Mechanism and Efficacy of Human Topoisomerase IB Inhibitors

Abstract: Human nuclear type IB topoisomerase (Top1) inhibitors are widely used and powerful anti-cancer agents. In this study, we introduce and validate a single-molecule supercoil relaxation assay as a molecular pharmacology tool for characterizing therapeutically relevant Top1 inhibitors. Using this assay, we determined the effects on Top1 supercoil relaxation activity of four Top1 inhibitors; three clinically relevant: camptothecin, LMP-400, LMP-776 (both indenoisoquinoline derivatives), and one natural product in preclinical development, lamellarin-D. Our results demonstrate that Top1 inhibitors have two distinct effects on Top1 activity: a decrease in supercoil relaxation rate and an increase in religation inhibition. The type and magnitude of the inhibition mode depend both on the specific inhibitor and on the topology of the DNA substrate. In general, the efficacy of inhibition is significantly higher with supercoiled than with relaxed DNA substrates. Comparing single-molecule inhibition with cell growth inhibition (IC50) measurements showed a correlation between the binding time of the Top1 inhibitors and their cytotoxic efficacy, independent of the mode of inhibition. This study demonstrates that the single-molecule supercoil relaxation assay is a sensitive method to elucidate the detailed mechanisms of Top1 inhibitors and is relevant for the cellular efficacy of Top1 inhibitors.

6,7-Dihydroxyisoindolin-1-one and 7,8-Dihydroxy-3,4-Dihydroisoquinolin- 1(2H)-one Based HIV-1 Integrase Inhibitors.

Abstract: Integrase (IN) is an essential viral enzyme required for HIV-1 replication, which has been targeted by anti-AIDS therapeutics. Integrase strand transfer inhibitors (INSTIs) represent a new class of antiretroviral agents developed for the treatment of HIV-1 infections. Important structural features that are shared by many INSTIs include a coplanar arrangement of three heteroatoms that chelate two catalytic Mg(2+) ions in the IN active site and a linked halophenyl ring that binds in the hydrophobic pocket formed by the complex of IN with viral DNA. We recently reported bicyclic 6,7-dihydroxyoxoisoindolin-1-one-based IN inhibitors. In the current study, we modified these inhibitors in three ways. First, we increased the spacer length between the metalchelating triad and the halophenyl group. Second, we replaced the indoline [5,6] bicycle with a fused dihydroxyisoquinolinones [6,6] ring system. Finally, we prepared bis-6,7-dihydroxyisoindolin-1-one-4-sulfonamides as dimeric HIV-1 IN inhibitors. These new analogues showed low micromolar inhibitory potency in in vitro HIV-1 integrase assays.

Abstract 2849: The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells

Abstract: Poly(ADP-ribose)polymerases (PARP) inhibitors work as catalytic inhibitors as well as PARP poisons by trapping PARP-DNA complexes (Murai et al., Cancer Res. 2012: 72:5588-99), and talazoparib is the most potent PARP inhibitor to date in terms of cytotoxicity and PARP trapping (Murai et al., Mol. Cancer Ther. 2014:13:433-43). Nevertheless, mining the NCI60 data shows that half of the 60 cell lines are tolerant to talazoparib. To elucidate the mechanisms of such tolerance, we searched for genes with expression levels correlated to talazoparib sensitivity using CellMiner (http://discover.nci.nih.gov/cellminer/). Schlafen 11 (SLFN11) turned out to be one of the most positively correlated genes whose expression is correlated with cellular sensitivity to talazoparib. Using genetically altered cells, we established that SLFN11 inactivation confers high resistance to talazoparib, revealing that SLFN11 inactivation, which is common in cancer cells (Zoppoli et al. PNAS 2014: 109:15030-5), is a novel mechanism of resistance for PARP inhibitors. Based on genetically engineered CRISPR/casp9 SLFN11-knockout cell lines and cell cycle analyses, we found that the parent SLFN11-proficient cells arrest in S phase without DNA replication, while SLFN11-knock out cells maintain their replication until entering G2 phase in the presence of talazoparib. Although ATR is the key regulator of intra-S phase check point, the addition of the ATR inhibitor (VE-821) (Josse et al., Cancer Res. 2014) does not rescue the replication arrest in SLFN11-proficient cells, suggesting the existence of a SLFN11-dependent replication arrest mechanism independently of ATR. On the other hand, the addition of the ATR inhibitor in SLFN11-knock out cells alters the arresting point from G2 to S phase, and eventually increases dead cells. This is probably because the prolonged replication block leads to the collapse of replication forks, resulting in DNA double-strand breaks. Consistent to these findings, the combination of the ATR inhibitor with talazoparib synergistically sensitizes SLFN11-knock out cells more efficiently than the parent SLFN11-proficient cells. Together, these results demonstrate that the combination of the ATR inhibitor with talazoparib can overcome the resistance of SLFN11-deficient cells to talazoparib. Citation Format: Junko Murai, Yves Pommier. The combination of the PARP inhibitor talazoparib (BMN 673) with the ATR inhibitor VE-821 overcomes the drug resistance of Schlafen 11-deficient cells. [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 2849. doi:10.1158/1538-7445.AM2015-2849

Production of Extrachromosomal MicroDNAs Is Linked to Mismatch Repair Pathways and

Abstract: MicroDNAs are <400-base extrachromosomal cir-cles found in mammalian cells. Tens of thousands ofmicroDNAs have been found in all tissue types,including sperm. MicroDNAs arise preferentiallyfrom areas with high gene density, GC content, andexon density from promoters with activating chro-matin modifications and in sperm from the 5