Size and Concentration of Cell-Free DNA Measured Directly from Blood Plasma, without Prior DNA Extraction.

Cell-free DNA in human blood plasma (cfDNA) is now widely used and studied as a biomarker for several physiological and pathological situations. In addition to genetic and epigenetic alterations that provide information about the presence and the nature of non-constitutive DNA in the body, cfDNA concentration and size distribution may potentially be independent biomarkers suitable for monitoring at-risk patients and therapy efficacy. Here, we describe a simple, in-line, method, which measures cfDNA concentration and size distribution from only a few microliters of plasma without the need to extract and/or concentrate the DNA prior to the analysis. This method is based on a dual hydrodynamic and electrokinetic actuation, adapted for samples containing salts and proteins such as biological fluids. The method provides analytical performances equivalent to those obtained after purification and concentration of cfDNA, with a precision of ∼1% for size features and of 10-20% for the concentrations of the different size fractions. We show that concentration and size distribution of cfDNA analyzed from plasma can differentiate advanced lung cancer patients from healthy controls. This simple and cost-effective method should facilitate further investigations into the potential clinical usefulness of cfDNA size profiling.

Productive HIV-1 infection of tissue macrophages by fusion with infected CD4+ T cells.

Macrophages are essential for HIV-1 pathogenesis and represent major viral reservoirs. Therefore, it is critical to understand macrophage infection, especially in tissue macrophages, which are widely infected in vivo, but poorly permissive to cell-free infection. Although cell-to-cell transmission of HIV-1 is a determinant mode of macrophage infection in vivo, how HIV-1 transfers toward macrophages remains elusive. Here, we demonstrate that fusion of infected CD4+ T lymphocytes with human macrophages leads to their efficient and productive infection. Importantly, several tissue macrophage populations undergo this heterotypic cell fusion, including synovial, placental, lung alveolar, and tonsil macrophages. We also find that this mode of infection is modulated by the macrophage polarization state. This fusion process engages a specific short-lived adhesion structure and is controlled by the CD81 tetraspanin, which activates RhoA/ROCK-dependent actomyosin contractility in macrophages. Our study provides important insights into the mechanisms underlying infection of tissue-resident macrophages, and establishment of persistent cellular reservoirs in patients.

Epigenetically regulated PCDHB15 impairs aggressiveness of metastatic melanoma cells.

The protocadherin proteins are cell adhesion molecules at the crossroad of signaling pathways playing a major role in neuronal development. It is now understood that their role as signaling hubs is not only important for the normal physiology of cells but also for the regulation of hallmarks of cancerogenesis. Importantly, protocadherins form a cluster of genes that are regulated by DNA methylation. We have identified for the first time that PCDHB15 gene is DNA-hypermethylated on its unique exon in the metastatic melanoma-derived cell lines and patients' metastases compared to primary tumors. This DNA hypermethylation silences the gene, and treatment with the DNA demethylating agent 5-aza-2'-deoxycytidine reinduces its expression. We explored the role of PCDHB15 in melanoma aggressiveness and showed that overexpression impairs invasiveness and aggregation of metastatic melanoma cells in vitro and formation of lung metastasis in vivo. These findings highlight important modifications of the methylation of the PCDHß genes in melanoma and support a functional role of PCDHB15 silencing in melanoma aggressiveness.

Tripartite split-GFP assay to identify selective intracellular nanobody that suppresses GTPase RHOA subfamily downstream signaling.

Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.

DNA methylome combined with chromosome cluster-oriented analysis provides an early signature for cutaneous melanoma aggressiveness.

Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients' outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

Early Steps of Resistance to Targeted Therapies in Non-Small-Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths among men and women worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective therapies for advanced non-small-cell lung cancer (NSCLC) patients harbouring EGFR-activating mutations, but are not curative due to the inevitable emergence of resistances. Recent in vitro studies suggest that resistance to EGFR-TKI may arise from a small population of drug-tolerant persister cells (DTP) through non-genetic reprogramming, by entering a reversible slow-to-non-proliferative state, before developing genetically derived resistances. Deciphering the molecular mechanisms governing the dynamics of the drug-tolerant state is therefore a priority to provide sustainable therapeutic solutions for patients. An increasing number of molecular mechanisms underlying DTP survival are being described, such as chromatin and epigenetic remodelling, the reactivation of anti-apoptotic/survival pathways, metabolic reprogramming, and interactions with their micro-environment. Here, we review and discuss the existing proposed mechanisms involved in the DTP state. We describe their biological features, molecular mechanisms of tolerance, and the therapeutic strategies that are tested to target the DTP.

Nanobody-Based Quantification of GTP-Bound RHO Conformation Reveals RHOA and RHOC Activation Independent from Their Total Expression in Breast Cancer.

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

Liquid Biopsy of Non-Plasma Body Fluids in Non-Small Cell Lung Cancer: Look Closer to the Tumor!

Liquid biopsy is a rapidly emerging field due to an increasing number of oncogenic drivers and a better understanding of resistance mechanisms to targeted therapies in non-small cell lung cancer (NSCLC). The sensitivity of the most widely used blood-based assays is, however, limited in particular in cases of low tumor volume where shed of tumor-derived material can be limited. A negative result thus requires biopsy confirmation using minimally invasive sampling procedures that can result in small specimens, which are often not suitable for genotyping. Liquid biopsy is not limited to plasma, and tumor DNA circulating in other body fluids such as urine, pleural fluid, cerebrospinal fluid, or cytology specimen-derived supernatant can be exploited. In comparison to cell blocks, these fluids in close contact to the tumor may contain a more abundant and less analytically demanding tumor DNA. In this review, we discuss the potential applications of circulating tumor DNA derived from cytology samples in NSCLC, from early stage (screening, nodule characterization) to metastatic disease.

Vγ9Vδ2 T Cells Activation Through Phosphoantigens Can Be Impaired by a RHOB Rerouting in Lung Cancer.

Vγ9Vδ2 T cells are known to be efficient anti-tumor effectors activated through phosphoantigens (PAg) that are naturally expressed by tumor cells or induced by amino bisphosphonates treatment. This PAg-activation which is TCR and butyrophilin BTN3A dependent can be modulated by NKG2D ligands, immune checkpoint ligands, adhesion molecules, and costimulatory molecules. This could explain the immune-resistance observed in certain clinical trials based on Vγ9Vδ2 T cells therapies. In NSCLC, encouraging responses were obtained with zoledronate administrations for 50% of patients. According to the in vivo results, we showed that the in vitro Vγ9Vδ2 T cell reactivity depends on the NSCLC cell line considered. If the PAg-pretreated KRAS mutated A549 is highly recognized and killed by Vγ9Vδ2 T cells, the EGFR mutated PC9 remains resistant to these killers despite a pre-treatment either with zoledronate or with exogenous BrHPP. The immune resistance of PC9 was shown not to be due to immune checkpoint ligands able to counterbalance NKG2D ligands or adhesion molecules such as ICAM-1 highly expressed by PC9. RHOB has been shown to be involved in the Vγ9Vδ2 TCR signaling against these NSCLC cell lines, in this study we therefore focused on its intracellular behavior. In comparison to a uniform distribution of RHOB in endosomes and at the plasma membrane in A549, the presence of large endosomal clusters of RHOB was visualized by a split-GFP system, suggesting that RHOB rerouting in the PC9 tumor cell could impair the reactivity of the immune response.

Current and future applications of liquid biopsy in nonsmall cell lung cancer from early to advanced stages.

Liquid biopsy refers to the analysis of any tumour-derived material circulating in the blood or any other body fluid. This concept is particularly relevant in lung cancer as the tumour is often difficult to reach and may need an invasive and potentially harmful procedure. Moreover, the multitude of anticancer drugs and their sequential use underline the importance of conducting an iterative assessment of tumour biology. Liquid biopsies can noninvasively detect any targetable genomic alteration and guide corresponding targeted therapy, in addition to monitoring response to treatment and exploring the genetic changes at resistance, overcoming spatial and temporal heterogeneity.In this article, we review the available data in the field, which suggest the potential of liquid biopsy in the area of lung cancer, with a particular focus on cell-free DNA and circulating tumour cells. We discuss their respective applications in patient selection and monitoring through targeted therapy, as well as immune checkpoint inhibitors. The current data and future applications of liquid biopsy in the early stage setting are also investigated.Liquid biopsy has the potential to help manage nonsmall cell lung cancer throughout all stages of lung cancer: screening, minimal residual disease detection to guide adjuvant treatment, early detection of relapse, systemic treatment initiation and monitoring of response (targeted or immune therapy), and resistance genotyping.

Notch inhibition overcomes resistance to tyrosine kinase inhibitors in EGFR-driven lung adenocarcinoma.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFRT790M and EGFRC797S tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3-dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor-resistant tumors, with EGFRT790M and EGFRC797S mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

Targeted sequencing of plasma cell-free DNA to predict response to PD1 inhibitors in advanced non-small cell lung cancer.

OBJECTIVES: Tumor mutational burden is an emerging biomarker of response to immune checkpoint inhibitors (ICI), whose clinical adoption is challenging. We hypothesized that targeting limited but relevant genetic alterations in plasma cell-free DNA along with early monitoring may non-invasively predict response to ICI in advanced non-small cell lung cancer (NSCLC). MATERIAL AND METHODS: Plasma samples from patients with progressive NSCLC collected before ICI initiation and at 1 month were profiled from responders (R: PFS > 6 months) and non-responders (NR: progressive disease at first evaluation) using amplicon sequencing of hotspots and coding regions from 36 genes. The molecular profile of ctDNA, and its early kinetics were analyzed. RESULTS: 97 patients were analyzed, of which 86 (39 R, 47 NR) were evaluable. Alterations in ctDNA were detectable in 67/86 baseline samples (78%). The detection of a targetable oncogenic driver was associated with a 2 months PFS. The presence of a PTEN or STK11 mutation was correlated with early progression (HR 8.9, p = 0.09 for PTEN, HR 4.7, p = 0.003 for STK11), while transversion mutations (Tv) in KRAS and TP53 predicted better outcomes (HR 0.36, p = 0.011 for TP53 Tv; HR 0.46, p = 0.11 for KRAS Tv). Patients with a low "immune score" (driver and/or PTEN or STK11 mutation and/or without KRAS or TP53 Tv) derived poor outcomes (median PFS 2 months), compared with patients with a high immune score (no driver, no PTEN or STK11 and with KRAS or TP53 Tv (median PFS 14 months, p = 0.0001, HR 2.96). Early changes in the ctDNA allele fraction (AF) of 65 specimens were correlated with clinical outcomes (14 months PFS if AF decreases vs. 2 months if AF increases, p < 0.0001). CONCLUSION: Targeted sequencing of plasma ctDNA and monitoring its early variations can predict response to ICI.

The RND1 Small GTPase: Main Functions and Emerging Role in Oncogenesis.

The Rho GTPase family can be classified into classic and atypical members. Classic members cycle between an inactive Guanosine DiPhosphate -bound state and an active Guanosine TriPhosphate-bound state. Atypical Rho GTPases, such as RND1, are predominantly in an active GTP-bound conformation. The role of classic members in oncogenesis has been the subject of numerous studies, while that of atypical members has been less explored. Besides the roles of RND1 in healthy tissues, recent data suggest that RND1 is involved in oncogenesis and response to cancer therapeutics. Here, we present the current knowledge on RND1 expression, subcellular localization, and functions in healthy tissues. Then, we review data showing that RND1 expression is dysregulated in tumors, the molecular mechanisms involved in this deregulation, and the role of RND1 in oncogenesis. For several aggressive tumors, RND1 presents the features of a tumor suppressor gene. In these tumors, low expression of RND1 is associated with a bad prognosis for the patients. Finally, we highlight that RND1 expression is induced by anticancer agents and modulates their response. Of note, RND1 mRNA levels in tumors could be used as a predictive marker of both patient prognosis and response to anticancer agents.

Hybrid QM/MM vs Pure MM Molecular Dynamics for Evaluating Water Distribution within p21N-ras and the Resulting GTP Electronic Density.

p21ras protein activity, regulated by GTP hydrolysis, constitutes an active field of research for the development of cancer targeted therapies that would concern ∼30% of human tumors to which specific mutations have been associated. Indeed, the catalyzing mechanisms provided by the protein environment during GTP hydrolysis and how they are impaired by specific mutations remain to be fully elucidated. In this article, we present results from molecular mechanics (MM) molecular dynamics (MD) simulations and density functional theory (DFT) calculations carried out for wild-type p21 N-ras and six Gln 61 mutants. In the first part, we present the water distribution within the active site of the wild-type protein according to MM MD. Significant differences are observed when comparing the results to the previous distribution assessed through quantum mechanics/molecular mechanics (QM/MM) MD. Such method-dependent results highlight the importance of accounting for the electrostatic coupling between the protein complex and the solvent molecules in identifying hydration sites. In the second part, we present the results from DFT calculations performed to determine the electronic distribution of the GTP ligand, considering the wild-type active site arrangement according to both classical and hybrid approaches. Only in the QM/MM-based configuration is the ligand electronic density similar to that of a GDP-like state observed experimentally. For this reason, in the last set of calculations carried out for p21 N-ras Gln 61 mutants, only the active site structural conformations obtained through hybrid MD are considered. Through the analysis of the GTP electronic density, we conclude that the wild-type active site arrangement according to QM/MM MD is closer to a catalytically efficient conformation of the protein than the arrangement according to MM MD. Hence, water distribution according to the hybrid approach must correspond to the optimal placement of solvent in the active site. Within all of the studied Gln 61 substituted proteins, p21ras major catalyzing effect, which consists of stabilizing a more GDP-like state, is lost.

Demethylation by low-dose 5-aza-2'-deoxycytidine impairs 3D melanoma invasion partially through miR-199a-3p expression revealing the role of this miR in melanoma.

BACKGROUND: Efficient treatments against metastatic melanoma dissemination are still lacking. Here, we report that low-cytotoxic concentrations of 5-aza-2'-deoxycytidine, a DNA demethylating agent, prevent in vitro 3D invasiveness of metastatic melanoma cells and reduce lung metastasis formation in vivo. RESULTS: We unravelled that this beneficial effect is in part due to MIR-199A2 re-expression by promoter demethylation. Alone, this miR showed an anti-invasive and anti-metastatic effect. Throughout integration of micro-RNA target prediction databases with transcriptomic analysis after 5-aza-2'-deoxycytidine treatments, we found that miR-199a-3p downregulates set of genes significantly involved in invasion/migration processes. In addition, analysis of data from melanoma patients showed a stage- and tissue type-dependent modulation of MIR-199A2 expression by DNA methylation. CONCLUSIONS: Thus, our data suggest that epigenetic- and/or miR-based therapeutic strategies can be relevant to limit metastatic dissemination of melanoma.

Cytoplasmic p27Kip1 promotes tumorigenesis via suppression of RhoB activity.

The cell cycle inhibitor p27Kip1 is a tumor suppressor via the inhibition of CDK complexes in the nucleus. However, p27 also plays other functions in the cell and may acquire oncogenic roles when located in the cytoplasm. Activation of oncogenic pathways such as Ras or PI3K/AKT causes the relocalization of p27 in the cytoplasm, where it can promote tumorigenesis by unclear mechanisms. Here, we investigated how cytoplasmic p27 participates in the development of non-small cell lung carcinomas. We provide molecular and genetic evidence that the oncogenic role of p27 is mediated, at least in part, by binding to and inhibiting the GTPase RhoB, which normally acts as a tumor suppressor in the lung. Genetically modified mice revealed that RhoB expression is preferentially lost in tumors in which p27 is absent and maintained in tumors expressing wild-type p27 or p27CK- , a mutant that cannot inhibit CDKs. Moreover, although the absence of RhoB promoted tumorigenesis in p27-/- animals, it had no effect in p27CK- knock-in mice, suggesting that cytoplasmic p27 may act as an oncogene, at least in part, by inhibiting the activity of RhoB. Finally, in a cohort of lung cancer patients, we identified a subset of tumors harboring cytoplasmic p27 in which RhoB expression is maintained and these characteristics were strongly associated with decreased patient survival. Thus, monitoring p27 localization and RhoB levels in non-small cell lung carcinoma patients appears to be a powerful prognostic marker for these tumors. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Early Circulating Tumour DNA Variations Predict Tumour Response in Melanoma Patients Treated with Immunotherapy.

Antibodies targeting immune checkpoints were recently approved for metastatic melanoma. However, not all patients will respond to the treatment and some will experience grade III-IV immune-related adverse events. Therefore, early identification of non-responder patients would greatly aid clinical practice. Detection of circulating tumour DNA (ctDNA) is a non-invasive approach to monitor tumour response. Digital droplet PCR was used to quantify BRAF and NRAS mutations in the plasma of patients with metastatic melanoma treated with immunotherapy. In 16 patients, ctDNA variations mirrored tumour response (p = 0.034) and ctDNA augmentation during follow-up detected tumour progression with 100% specificity. In 13 patients, early ctDNA variation was associated with clinician decision at first evaluation (p = 0.0046), and early ctDNA increase with shorter progression-free survival (median 21 vs. 145 days; p = 0.001). Monitoring ctDNA variations early during immunotherapy may help clinicians rapidly to discriminate non-responder patients, allow early adaptation of therapeutic strategies, and reduce exposure to ineffective, expensive treatment.

Water Distribution within Wild-Type NRas Protein and Q61 Mutants during Unrestrained QM/MM Dynamics.

Point mutations in p21ras are associated with ∼30% of human tumors by disrupting its GTP hydrolysis cycle, which is critical to its molecular switch function in cellular signaling pathways. In this work, we investigate the impact of Gln 61 substitutions in the structure of the p21N-ras active site and particularly focus on water reorganization around GTP, which appears to be crucial to evaluate favorable and unfavorable hydration sites for hydrolysis. The NRas-GTP complex is analyzed using a hybrid quantum mechanics/molecular mechanics approach, treating for the first time to our knowledge transient water molecules at the ab initio level and leading to results that account for the electrostatic coupling between the protein complex and the solvent. We show that for the wild-type protein, water molecules are found around the GTP γ-phosphate group, forming an arch extended from residues 12 to 35. Two density peaks are observed, supporting previous results that suggest the presence of two water molecules in the active site, one in the vicinity of residue 35 and a second one stabilized by hydrogen bonds formed with nitrogen backbone atoms of residues 12 and 60. The structural changes observed in NRas Gln 61 mutants result in the drastic delocalization of water molecules that we discuss. In mutants Q61H and Q61K, for which water distribution is overlocalized next to residue 60, the second density peak supports the hypothesis of a second water molecule. We also conclude that Gly 60 indirectly participates in GTP hydrolysis by correctly positioning transient water molecules in the protein complex and that Gln 61 has an indirect steric effect in stabilizing the preorganized catalytic site.

PARP-1-dependent RND1 transcription induced by topoisomerase I cleavage complexes confers cellular resistance to camptothecin.

RHO GTPases regulate essential functions such as the organization of the actin cytoskeleton. The classic members cycle between an active GTP-bound and an inactive GDP-bound conformation whereas atypical members are predominantly GTP-bound. Besides their well-established role, the classic RHO GTPases RHOB and RAC1, are rapidly induced and/or activated by genotoxic stress and contribute to the DNA damage response. Here we used camptothecin, a selective topoisomerase I (TOP1) inhibitor that stabilizes TOP1 cleavage complexes (TOP1cc), to search for other potential early DNA damage-inducible RHO GTPase genes. We identified that an atypical RHO GTPase, RND1, is rapidly induced by camptothecin. RND1 induction is closely associated with the presence of TOP1cc induced by camptothecin or by DNA lesions that elevate TOP1cc levels such as UV and hydrogen peroxide. We further demonstrated that camptothecin increases RND1 gene transcription and mRNA stability. Camptothecin also increases poly(ADP-ribose) polymerase 1 (PARP-1) activity, whose inhibition reduces RND1 transcription. In addition, overexpression of RND1 increases PARP-1, suggesting a cross-talk between PARP-1 and RND1. Finally, RND1 protects cells against camptothecin-induced apoptosis, and hence favors cellular resistance to camptothecin. Together, these findings highlight RND1 as an atypical RHO GTPase early induced by TOP1cc, and show that the TOP1cc-PARP-1-RND1 pathway protects cells against apoptosis induced by camptothecin.