Céline Auzanneau

New Topoisomerase I mutations are associated with resistance to camptothecin

BackgroundTopoisomerase I (TOP1) is a nuclear enzyme that catalyzes the relaxation of supercoiled DNA during DNA replication and transcription. TOP1 is the molecular target of camptothecin and related drugs such as irinotecan and SN38 (irinotecans active metabolite). Irinotecan is widely used as an anti-cancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance.MethodsWe previously established several SN38 resistant HCT116-derived clones to study the mechanisms underlying resistance to SN38. Here, we investigated whether resistance to SN38 in these cell lines could be linked to the presence of TOP1 mutations and changes in its expression and activity. Functional analyses were performed on these cell lines challenged with SN38 and we specifically monitored the double strands breaks with γH2AX staining and replication activity with molecular combing.ResultsIn SN38 resistant HCT116 clones we identified three new TOP1 mutations, which are located in the core subdomain III (p.R621H and p.L617I) and in the linker domain (p.E710G) and are packed together at the interface between these two domains. The presence of these TOP1 mutations in SN38 resistant HCT116 cells did not modify TOP1 expression or intrinsic activity. Conversely, following challenge with SN38, we observed a decrease of TOP1-DNA cleavage complexes and a reduction in double-stranded break formation). In addition, we showed that SN38 resistant HCT116 cells present a strong decrease in the SN38-dependent asymmetry of replication forks that is characteristic of SN38 sensitive HCT116 cells.ConclusionsThese results indicate that the TOP1 mutations are involved in the development of SN38 resistance. We hypothesize that p.L617, p.R621 and p.E710 TOP1 residues are important for the functionality of the linker and that mutation of one of these residues is sufficient to alter or modulate its flexibility. Consequently, linker fluctuations could have an impact on SN38 binding by reducing the enzyme affinity for the drug.

The polyphenolic ellagitannin vescalagin acts as a preferential catalytic inhibitor of the α isoform of human DNA topoisomerase II.

Polyphenolic ellagitannins are natural compounds that are often associated with the therapeutic activity of plant extracts used in traditional medicine. They display cancer-preventing activity in animal models by a mechanism that remains unclear. Potential targets have been proposed, including DNA topoisomerases II (Top2). Top2α and Top2β, the two isoforms of the human Top2, play a crucial role in the regulation of replication, transcription, and chromosome segregation. They are the target of anticancer agents used in the clinic such as anthracyclines (e.g., doxorubicin) or the epipodophyllotoxin etoposide. It was recently shown that the antitumor activity of etoposide was due primarily to the inhibition of Top2α, whereas inhibition of Top2β was responsible for the development of secondary malignancies, pointing to the need for more selective Top2α inhibitors. Here, we show that the polyphenolic ellagitannin vescalagin preferentially inhibits the decatenation activity of Top2α in vitro, by a redox-independent mechanism. In CEM cells, we also show that transient small interfering RNA-mediated down-regulation of Top2α but not of Top2β conferred a resistance to vescalagin, indicating that the α isoform is a preferential target. We further confirmed that Top2α inhibition was due to a catalytic inhibition of the enzyme because it did not induce DNA double-strand breaks in CEM-treated cells but prevented the formation of Top2α- rather than Top2β-DNA covalent complexes induced by etoposide. To our knowledge, vescalagin is the first example of a catalytic inhibitor for which cytotoxicity is due, at least in part, to the preferential inhibition of Top2α.

DNA Topoisomerase I and Illegitimate Recombination

DNA topoisomerase IB (Top1) is a ubiquitous nuclear enzyme whose main role is to remove torsional tensions associated with transcription or replication by introducing transient single-strand breaks in duplex DNA. Once supercoils are suppressed, DNA breaks are rapidly resealed during the religation step of the Top1 reaction. The striking similarity between Top1-mediated religation and the strand transferase activity of various recombinases have suggested that Top1 could be involved in DNA recombination and contribute to the maintenance of genomic integrity. Moreover, Top1 is routinely used as a reagent for molecular cloning (Topo® PCR cloning). In this chapter, we will review the experimental evidences suggesting the potential role of Top1 in illegitimate recombination either via its strand transferase activity, or independently of its religation activity via the regulation of other cellular mechanisms such as transcription or DNA repair.

Targeting ERBB2 mutations in solid tumors: biological and clinical implications

Preclinical data have shown that ERBB2 activating mutations are responsive to HER2 tyrosine kinase inhibitors. The aim of this study is to characterize the landscape of ERBB2 mutations in solid tumors and the potential efficacy of ERBB2 targeting.We analyzed the next-generation sequencing results from 17,878 patients with solid tumors and reported the outcome of 4 patients with advanced ERBB2-mutated tumors treated with a combination of trastuzumab and lapatinib.ERBB2 mutations occurred in 510 patients (2.85%). The tumor types with the highest incidence of ERBB2 mutations were the following: bladder (16.6%), small bowel (8.6%), ampullar (6.5%), skin non-melanoma (6.1%), and cervical cancer (5.5%). 49.4% (n = 282) were known as activating mutations. ERBB2 mutation was not mutually exclusive of ERBB2 amplification which occurred in up to 10% of cases. PI3KCA activating mutations were associated with ERBB2 mutations in 12.4% of cases mainly in breast and lung cancer. Four patients (endometrial, colorectal, cholangiocarcinoma, and adenosarcoma of the uterus) were treated with a combination of trastuzumab and lapatinib. All of them experienced tumor shrinkage resulting in stable disease in three cases and partial response in one case. One patient developed secondary resistance. Sequencing of the progressing metastasis allowed the identification of the ERBB2 L869R mutation previously associated with resistance to lapatinib in vitro.These results support further clinical investigation aiming to demonstrate that ERBB2-mutational driven therapy can improve patient care irrespective of histology.

Abstract C48: BIP (Bergonie Institute profiling) program: Fighting cancer by matching molecular alterations and drugs in early phase trials

Background: BIP is an institution-wide permanent screening program started in 2014 to identify patients (pts) referred to Institut Bergonie (Bordeaux, France) with somatic alterations that can be matched to targeted therapies in early phase clinical trials. Methods: Pts with advanced solid tumors and with ECOG performance status ≤ 2 were eligible. Tumor DNA was isolated from a FFPE archived sample when available or from a fresh tumor biopsy. DNA analysis was performed by next generation sequencing (NGS) using a panel of up to 287 genes and by comparative genomic hybridization (CGH). Results for each patient were discussed during a weekly multidsicplinary molecular tumor board in order to assess the eligibility of the patient to early phase clinical trials. Results: From Jan 1 2014 to June 30 2015, 542 pts were enrolled with median 2 prior treatments for advanced disease (range 0-9). The main tumor types were: lung (19.2%), colorectal (16.2%), breast (13.3%), ovarian (11%), and sarcomas (10%). Median age was 61 years (range 18-84). In 28 cases (5%) molecular analysis failed mainly because of insufficient tissue. The median time from first referral to reporting was 9 weeks (range 1-36). The 20 genes most frequently altered were TP53, CDKN2A,PTEN, CDKN2B, PIK3CA, MYC, ARID1A, KRAS, RB1, EGFR, ERBB2, FGFR1, APC,RICTOR,ZNF703,ATM,BRAF,NF1,FGFR3 and CCND1. Among the patients included between Jan 1 2014 and January 31 2015 (n = 286), 176 patients (68%) of patients had at least one genetic alteration that was considered actionable by the molecular tumor board. 85 patients (29.7%) were included in a clinical trial with a median delay of 17 weeks between first referral and date of treatment onset. The treatment was matched with the tumor profile in 49 cases (17%). The main reasons for non-inclusion in a clinical trial despite the identification of an actionable mutation were: non progressive disease on the current line of treatment (31.5%), general status deterioration (26%), death (13%), clinical trial not available (13%), screening failure (6.5%), lost of follow-up (5.5%), and patient refusal (4.5%). 79 patients were evaluable for response according to RECIST 1.1. The disease control rate (objective response + stable disease) was 47% for patients included in clinical trials matched with the tumor profile versus 53% (p = 0.9) for the group of patients included in other clinical trials The median progression-free survival was 3.6 months (95 CI 1.8-5.3) versus 3.6 months (95 CI 0.9-6.3) (p = 0.5). Conclusions: Extensive molecular profiling by using high-throughput techniques is feasible in routine practice, allow identification of actionable mutations in the majority of cases and can be used to enroll patients in early phase trials matched to their tumor genotype. Citation Format: MAUD TOULMONDE, THOMAS GRELLETY, CELINE AUZANNEAU, YEC9HAN LAIZET, KEVIN TRAN, ANNE FLOQUET, DELPHINE GARBAY, JACQUES ROBERT, ISABELLE HOSTEIN, ISABELLE SOUBEYRAN, ANTOINE ITALIANO. BIP (Bergonie Institute profiling) program: Fighting cancer by matching molecular alterations and drugs in early phase trials. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C48.

Conception, mise en place et fonctionnement d’une RCP dédiée aux essais précoces : l’exemple de l’Institut Bergonié

Nous rapportons l’experience de l’Institut Bergonie dans la mise en place en routine d’un programme de cartographie genomique tumorale au sein d’une reunion de concertation pluridisciplinaire (RCP) dediee. Nous discutons egalement les nombreux defis emergeant dans l’acces a la medecine de precision en oncologie.

Abstract 2200: Relationship between cytochrome P450 1B1 (CYP1B1) and head and neck cancer cells proliferation, dissemination and chemosensitivity to anticancer drugs.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Contrasting to other cytochromes P450, CYP1B1 is essentially present in tumour tissue and has been suspected to play a role in oncogenesis and drug resistance. We have recently shown that a CYP1B1 single nucleotide gene polymorphism (rs1056836, 1697 G>C, V432L) was significantly associated with the cytotoxicity of DNA-damaging anticancer drugs in two independent panels of tumour cell lines, the NCI-60 panel and the JFCR-45 panel. In order to identify the molecular mechanisms involved in the role of CYP1B1 polymorphism in anticancer drug resistance, we selected head-and-neck cancer cell lines with no basal expression of CYP1B1 due to promoter hypermethylation and generated by lentiviral infection isogenic variants differing only by this single nucleotide in the CYP1B1 gene. Cell proliferation was strongly enhanced in cells re-expressing CYP1B1 and further increased in cells harbouring the C/C genotype (C/C cells) as compared to those harbouring the G/G genotype (G/G cells). The cytotoxicity of a panel of anticancer drugs from all drug classes was evaluated on these established cell lines. Significantly higher IC50s of most of them (excepted oxaliplatin and gemcitabine) were noticed for C/C cells as compared to G/G cells in agreement with what was observed in the NCI and JFCR panels. Migration capacity of these cell lines was studied using the scratch wound assay and proteins involved in cell adhesion were evaluated by Western blotting. Cells re-expressing CYP1B1 were able to migrate significantly more rapidly than original cells and this was further enhanced in C/C cells as compared to G/G cells. E-cadherin expression was decreased in C/C cells while vimentin expression was increased in G/G cells. The clinical consequences of the CYP1B1 L432V gene polymorphism were studied in a prospective population of 123 head-and-neck cancer patients treated by chemotherapy associated with cetuximab in the palliative setting, either with locally advanced or with metastatic disease. There were 48 C/C patients, 40 heterozygotes C/G and 35 G/G patients, giving an allele frequency of 45%. No differences in progression rates as a function of the polymorphism were noticed. Overall median survival (OMS) was significantly worse in C/C patients (6.6 months) than in G/G patients (13.2 months), heterozygotes falling in between (9.8 months) (p = 0.017). Taking into account only the metastatic patients (47 patients) revealed more profound differences between C/C patients (OMS = 3.8 months) and G/G patients (OMS not reached), with heterozygotes in between (OMS = 12.0 months) (p = 0.0003). CYP1B1 V432L gene polymorphism appears as a strong determinant of survival of patients with advanced head-and-neck cancer and treated by chemotherapy associated with cetuximab. Citation Format: Valerie Le Morvan, Celine Auzanneau, Alban Pasquies, Mathilde Brault, Jacques Robert, Amelie Lansiaux. Relationship between cytochrome P450 1B1 (CYP1B1) and head and neck cancer cells proliferation, dissemination and chemosensitivity to anticancer drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2200. doi:10.1158/1538-7445.AM2013-2200