The resolution of DNA interstrand crosslinks (ICLs) requires a complex interplay between several processes of DNA metabolism, including the Fanconi anemia (FA) pathway and homologous recombination (HR). FANCD2 monoubiquitination and CtIP-dependent DNA-end resection represent key events in FA and HR activation, respectively, but very little is known about their functional relationship. Here, we show that CtIP physically interacts with both FANCD2 and ubiquitin and that monoubiquitinated FANCD2 tethers CtIP to damaged chromatin, which helps channel DNA double-strand breaks generated during ICL processing into the HR pathway. Consequently, CtIP mutants defective in FANCD2 binding fail to associate with damaged chromatin, which leads to increased levels of nonhomologous end-joining activity and ICL hypersensitivity. Interestingly, we also observe that CtIP depletion aggravates the genomic instability in FANCD2-deficient cells. Thus, our data indicate that FANCD2 primes CtIP-dependent resection during HR after ICL induction but that CtIP helps prevent illegitimate recombination in FA cells.

Highlights d CtIP protects reversed forks from nucleolytic degradation d CtIP prevents DNA2-dependent over-resection of nascent strands d CtIP nuclease mutants are defective in fork protection d CtIP-mediated fork protection synergizes with BRCA1, not BRCA2

Background: Extensive DNA sequencing has led to an unprecedented view of the diversity of individual genomes and their evolution among patients with clear cell renal cell carcinoma (ccRCC). Objective: To understand subclonal architecture and dynamics of patient-derived two-dimensional (2D) and three-dimensional (3D) ccRCC models in vitro, in order to determine whether they mirror ccRCC inter-and intratumor heterogeneity. Design, setting, and participants: We have established a comprehensive platform of living renal cancer cell models from ccRCC surgical specimens. Outcome measurements and statistical analysis: We confirmed the concordance of 2D and 3D patient-derived cell (PDC) models with the original tumor tissue in terms of histology, biomarker expression, cancer driver mutations, and copy number alterations. We addressed inter-and intrapatient heterogeneity by analyzing clonal dynamics during serial passaging. Results and limitations: In-depth genetic characterization verified the presence of heterogeneous cell populations, and revealed a high degree of similarity between subclonal compositions of monolayer and organoid cell cultures and the corresponding parental ccRCCs. Clonal dynamics were evident during serial passaging of cells in vitro, suggesting that PDC cultures can offer insights into evolutionary potential and treatment susceptibility of ccRCC subclones in vivo. Proof-of-concept drug profiling using selected ccRCC-targeted therapy agents highlighted patient-specific vulnerabilities in PDC models that could not be anticipated by interrogating commercially available cell lines. Conclusions: We demonstrate that PDC models mirror inter-and intratumor heterogeneity of ccRCC in vitro. Based on our findings, we envision that the use of these models will advance our understanding of the trajectories that cause genetic diversity and their consequences for treatment on an individual level. Patient summary: In this study, we developed two-and three-dimensional patient-derived models from clear cell renal cell carcinoma (ccRCC) as "mini-tumors in a dish." We show that these cell models retain important features of the human ccRCCs such as the profound tumor heterogeneity, thus highlighting their importance for cancer research and precision medicine.

Genomic instability, a hallmark of almost all human cancers, drives both carcinogenesis and resistance to therapeutic interventions. Pivotal to the ability of a cell to maintain genome integrity are mechanisms that signal and repair deoxyribonucleic acid (DNA) double-strand breaks (DSBs), one of the most deleterious lesions induced by ionising radiation and various DNA-damaging chemicals. On the other hand, many current therapeutic regimens that effectively kill cancer cells are based on the induction of excessive DSBs. However, these drugs often lack selectivity for tumour cells, which results in severe side effects for the patients, thus compromising their therapeutic potential. Therefore, the development of novel tumour-specific treatment strategies is required. Unlike normal cells, however, cancer cells are often characterised by abnormalities in the DNA damage response including defects in cell cycle checkpoints and/or DNA repair, rendering them particularly sensitive to the induction of DSBs. Therefore, new anticancer agents designed to exploit these vulnerabilities are becoming promising drugs for enhancing the specificity and efficacy of future cancer therapies. Here, we summarise the latest preclinical and clinical developments in cancer therapy based on the current knowledge of DSB signalling and repair, with a special focus on the combination of small molecule inhibitors with synthetic lethality approaches.

Activating mutations of the class III receptor tyrosine kinase FLT3 are the most frequent molecular aberration in acute myeloid leukemia (AML). Mutant FLT3 accelerates proliferation, suppresses apoptosis, and correlates with poor prognosis. Therefore, it is a promising therapeutic target. Here, we show that RNA interference against FLT3 with an internal tandem duplication (FLT3-ITD) potentiates the efficacy of the histone deacetylase inhibitor (HDACi) panobinostat (LBH589) against AML cells expressing FLT3-ITD. Similar to RNA interference, tyrosine kinase inhibitors (TKI; AC220/cpd.102/PKC412) in combination with LBH589 exhibit superior activity against AML cells. Median dose-effect analyses of drug-induced apoptosis rates of AML cells (MV4-11 and MOLM-13) revealed combination index (CI) values indicating strong synergism. AC220, the most potent and FLT3-specific TKI, shows highest synergism with LBH589 in the low nanomolar range. A 4-hour exposure to LBH589 þ AC220 already generates more than 50% apoptosis after 24 hours. Different cell lines lacking FLT3-ITD as well as normal peripheral blood mononuclear cells are not significantly affected by LBH589 þ TKI, showing the specificity of this treatment regimen. Immunoblot analyses show that LBH589 þ TKI induce apoptosis via degradation of FLT3-ITD and its prosurvival target STAT5. Previously, we showed the LBH589-induced proteasomal degradation of FLT3-ITD. Here, we show that activated caspase-3 also contributes to the degradation of FLT3-ITD and that STAT5 is a direct target of this protease. Our data strongly emphasize HDACi/TKI drug combinations as promising modality for the treatment of FLT3-ITD-positive AMLs. Mol Cancer Ther; 11(11); 2373-83. Ó2012 AACR.

Histone acetylation and methylation are linked to a variety of nuclear activities, most notably transcriptional regulation. Both synergistic and antagonistic relationships between these two modifications have been reported in different systems. Here we show that the budding yeast histone H4 arginine 3 (R3) methyltransferase Hmt1p binds acetylated histones H3 and H4, and importantly, that acetylated H4 is a significantly better methylation substrate for Hmt1p. Kinetic studies show that acetylation at any of the four acetylatable lysine residues of histone H4 results in more efficient methylation. Among the four, K8 acetylation imposes the strongest effect on reducing K M , consistent with the observed acetylation-stimulated interaction. In vivo, hmt1! cells rescue the transcriptional defect caused by GCN5 deletion, indicating that one of the functions of Gcn5p is to neutralize the negative effect of Hmt1p. Mutating either K8 or R3 to alanine causes similar growth defects in selective histone and gcn5 mutant background, suggesting that these two residues function in the same pathway for optimal vegetative growth. Together, these results reveal functional connection between histone acetylation, methylation, and two of the responsible enzymes, Gcn5p and Hmt1p.

Biorespositories of formalin-fixed and paraffin-embedded (FFPE) or fresh frozen human tissues from malignant diseases generated as integral part of the diagnostic workup in many pathology departments have been pivotal resources for translational cancer studies. However, such tissue biobanks have traditionally contained only non-viable specimens and thus cannot enable functional assays for the discovery and validation of therapeutic targets or the assessment of drug responses and resistance to treatment. To overcome these limitations, we have developed a next-generation comprehensive biobanking platform that includes the generation of patient-derived in vitro cell models from colorectal, pancreatic and kidney cancers among others. As such patient-derived cell (PDC) models retain important features of the original human tumors, they have emerged as relevant tools for more dynamic clinical and experimental analyses of cancer. Here, we describe details of the complex processes of acquisition and processing of patient-derived samples, propagation, annotation, characterization and distribution of resulting cell models and emphasize the requirements of quality assurance, organizational considerations and investment into resources. Taken together, we show how clinical tissue collections can be taken to the next level thus promising major new opportunities for understanding and treating cancer in the context of precision medicine.

Clear cell renal cell carcinoma (ccRCC) displays a highly varying clinical progression, from slow growing localized tumors to very aggressive metastatic disease (mRCC). Almost a third of all patients with ccRCC show metastatic dissemination at presentation while another third develop metastasis during the course of the disease. Survival rates of mRCC patients remain low despite the development of novel targeted treatment regimens. Biomarkers indicating disease progression could help to define its aggressive potential and thus guide patient management. However, molecular markers that can reliably assess metastatic dissemination and disease recurrence in ccRCC have not been recommended for clinical practice to date. Liquid biopsies could provide an attractive and non-invasive method to determine the risk of recurrence or metastatic dissemination during follow-up and thus assist the search for surveillance biomarkers in ccRCC tumors. A wide spectrum of circulating molecules have already shown considerable potential for ccRCC diagnosis and prognostication. In this review, we outline state of the art of the key circulating analytes such as cfDNA, cfRNA, proteins, and exosomes that may serve as biomarkers for the longitudinal monitoring of ccRCC progression to metastasis. Moreover, we address some of the prevailing limitations in the past approaches and present promising adoptable technologies that could help to pursue the implementation of liquid biopsies as a prognostic tool for mRCC.