535. Increasing Accuracy and Precision of Vector Integration Site Identification of Sequencing Reads With a New Bioinformatics Framework

In hematopoietic stem cell (HSC) gene therapy (GT) applications patients are transplanted with autologuos HSCs that have been ex-vivo genetically modified with integration competent vectors to express a therapeutic transgene. Specific PCR techniques coupled to next generation sequencing and bioinformatics analysis allow the high throughput retrieval, sequencing and mapping of proviral/genomic DNA junctions present in the blood and bone marrow derived cell populations sampled at different time points after therapy. The increase in sequences available for IS mapping is accompanied by an increase in false positives derived by sequencing errors or sequencing read parsing and mapping on the reference genome. In particular, by analyzing IS datasets form vector marked human and mouse tumor cells, clones with defined integration sites and GT patients, we observed that when multiple sequences arising from the same IS are aligned on the reference genome >10% mapped near (+/- 4 bases) the true insertion site. Without correction, these misaligned sequences not only result in an overestimation of the overall number of IS but in some cases also in the generation of false common insertion sites, worrisome hallmarks of insertional mutagenesis. To mitigate this issue we and others, based on empirical observations, merge sequencing reads mapping within +/- 3 bp into a single IS. Although this adjustment reduces the impact of the "wobbling" around the true ISs, a dedicated method and model is still missing.To further increase the accuracy of genomic positioning of sequencing reads we developed a new bioinformatics framework as post-processing plugin for pipelines that correctly partitions sequencing reads in a given genomic position by considering the relative abundance and distribution of each sequence cluster using local modes and Gaussian scores through an adaptive approach that varies the parameters of the Gaussian curve and proposes different solutions. To chose the best solution, the algorithm first evaluates each solution by exploiting 100 simulations of the input reads and then selects the resulting best solution using the Kolmogorov-Smirnov test. The simulation step is designed to test the mappability of the IS genomic interval and to quantify the impact of the observed nucleotide variations of the reads with respect to the reference genome (PCR artifacts or real genomic differences) that may lead to different mapping results that justify a larger span of the mapped reads surrounding the putative IS. The algorithm returns the list of IS and relative number of reads with the p-value of the best solution.We performed 3 ad-hoc in vitro experiments on a cell clone with 6 known IS in which we measured the precision of IS placement obtaining an average of 100% with our new method whereas <30% using our previous method based on a rigid sliding window approach of 4 bp. We applied our new approach to our clinical trial datasets obtaining improvements in IS genomic placement and overestimation with a reduction of potential false IS of 3% without changing the biological results

537. New Graph-Based Algorithm for Comprehensive Identification and Tracking Retroviral Integration Sites

Vector integration sites (IS) in hematopoietic stem cell (HSC) gene therapy (GT) applications are stable genetic marks, distinctive for each independent cell clone and its progeny. The characterization of IS allows to identify each cell clone and individually track its fate in different tissues or cell lineages and during time, and is required for assessing the safety and efficacy of the treatment. Bioinformatics pipelines for IS detection used in GT identify the sequence reads mapping in the same genomic position of the reference genome as a single IS but discard those ambiguously mapped in multiple genomic regions. The loss of such significant portion of patients' IS may hide potential malignant events thus reducing the reliability of IS studies. We developed a novel tool that is able to accurately identify IS in any genomic region even if composed by repetitive genomic sequences. Our approach exploits an initial genome free analysis of sequencing reads by creating an undirected graph in which nodes are the input sequences and edges represent valid alignments (over a specific identity threshold) between pairs of nodes. Through the analysis and decomposition of the graph, the method identifies indivisible subgraphs of sequences (clusters), each of them corresponding to an IS. Once extracted the consensus sequence of the clusters and aligned on the reference genome, we collect the alignment results and the annotation labels from RepeatMasker. By combining the set of genomic coordinates and the annotation labels, the method retraces the initial sequence graph, statistically validates the clusters through permutation test and produces the final list of IS. We tested the reliability of our tool on 3 IS datasets generated from simulated sequencing reads with incremental rate of nucleotide variations (0%, 0.25% and 0.5%) and real data from a cell line with known IS and we compared out tool to VISPA and UClust, used for GT studies. In the simulated datasets our tool demonstrated precision and recall ranging 0.85-0.97 and 0.88-0.99 respectively, producing the aggregate F-score ranging 0.86-0.98 which resulted higher than VISPA and UClust. In the experimental case of sequences from LAM-PCR products, our tool and VISPA were able to identify all the 6 known ISs for >98% of the reads produced, while UClust identified only 5 out 6 ISs. We then used our tool to reanalyze the sequencing reads of our GT clinical trial for Metachromatic Leukodystrophy (MLD) completing the hidden portion of IS. The overall number of ISs, sequencing reads and estimated actively re-populating HSCs was increased by an average fold ~1.5 with respect the previously published data obtained through VISPA whereas the diversity index of the population did not change and no aberrant clones in repeats occurred. Our tool addresses and solves important open issues in retroviral IS identification and clonal tracking, allowing the generation of a comprehensive repertoire of IS

674 insertional mutagenesis to identify mechanisms of cetuximab resistance in colorectal cancer

Anti-cancer drugs designed to target specific molecular pathways have shown an excellent therapeutic potential but also very poor long-term durability of tumor responses, mainly due to the outbreak of resistant clones among the residual neoplastic cell population. For that reason, understanding the molecular mechanisms underlying the onset of anti-cancer drug resistance (ACDR) is one of the major goals of clinical research. ACDR has been widely studied by DNA/RNA sequencing of primary human samples and several culprits identified. We have previously developed an approach based on lentiviral vector (LV)-induced insertional mutagenesis that allowed to identify the genes involved in lapatinib and erlotinib resistance on HER2+ human breast cancer cell lines and EGFR+ pancreatic cell line respectively. Here we took advantage of this platform to investigate ACDR genes in colorectal cancer (CRC). Cetuximab, anti-EGFR monoclonal antibody, is used as first line therapy in metastatic CRC, which results in prolonged survival of treated patients. However, nearly all patients relapse due to ACDR. We thus selected CRC cells sensitive to cetuximab deriving either from five microsatellite stable cell lines or from eight Patient Derived Xenografts (PDX), primary human CRC cells implanted subcutaneously into immunodeficient mice (NSG). To induce insertional mutagenesis we generated a luciferase-expressing LV harboring the SFFV enhancer/promoter in the long terminal repeats able to perturb the expression of genes nearby the integration site. As control, we used a non-genotoxic SIN-LV. We set up a collagenase IV-based disaggregation protocol that allows single-cell suspension and a serum-free culture condition to maintain the stemness of in vitro cultured cells. This protocol allowed to efficiently disaggregate and expand CRC cells in vitro as well as reach a LV copy number per cell ranging from 0.25 to 5.6. Luciferase gene expression was stable and allowed live-animal monitoring for up to 30 weeks after transplant. CRC-0069 and -0077 PDXs and NCI-H508 and HDC82 cell lines were transduced ex vivo and kept in vitro and/or transplanted in NSG mice. After in vitro or in vivo expansion of the transduced CRCs cetuximab treatment was applied. After an initial shrinking of the tumor mass in mice we observed ACDR in 3 out of 10 mice transplanted with NCI-H508 cells transduced with SFFV-LV and in none of the controls. Genomic DNA from resistant cells is being used for insertion site (IS) analysis to identify common IS, ACDR gene candidates. IS obtained from SIN-LV groups will be used to filter LV integration biases, whereas IS from SFFV-LV transduced cells but not treated with cetuximab will be used to filter mutations that provide a proliferative advantage unrelated to cetuximab treatment. We will validate the most promising candidates by LV-mediated overexpression and knockdown techniques. This approach could pave the way to perform insertional mutagenesis-based forward genetics studies on primary human samples

3. Safety Assessment of SIN LVs Harboring Chromatin Insulators in the Sensitive Cdkn2a-/- In Vivo Genotoxicity Assay Show Enhancer-Blocking Activity of Specific Insulator Sequences

Chromatin insulators (CI) have been proposed as safety features to increase the safety of self-inactivating (SIN) lentiviral vectors (LV) for gene therapy applications.By taking advantage of an in vivo genotoxicity assay based on the systemic injection of LVs in newborn tumor-prone Cdkn2a-/- mice we were able to measure vector-induced genotoxicity as an accelerated tumor onset that was proportional to the genotoxic potential of the tested LV. Importantly, we took advantage of integration sites (IS) analysis to qualitatively characterize CI that were shown by other in vitro and ex vivo studies to function as insulators. Recently we showed for the first time that a CAAT-box binding Nuclear factor 1 (CTF/NF1)-based CI, when cloned in the LTRs of a SIN.LV with a strong SFFV enhancer-promoter in internal position, significantly reduced the frequency of tumors harboring integrations activating Map3k8 oncogene accompanied by a marked skewing towards tumors harboring inactivating insertions targeting Pten.Here by using this stringent in vivo genotoxicity assay and IS analysis in tumors we expanded our studies towards other CI sequences whose function is regulated by the binding of the CCCTC-binding factor (CTCF), the best characterized insulator protein in vertebrates.Each CTCF-based insulating cassette was cloned in the LTRs of a LV construct containing the SFFV promoter in internal position (CTCF.SIN.LVs) and injected in Cdkn2a-/- mice. Interestingly, mice treated with some of the CTCF.SIN.LVs tested displayed an increased median survival time (ranging from 193.5 to 214 days) compared to mice treated with the uninsulated parental SIN.LV (186 days). Importantly, our preliminary IS analysis in tumors (881 IS) showed that two CTCF.SIN.LVs did not target Map3k8 oncogene while Pten was often disrupted by exonic insertions, an escape genotoxicity mechanism on which CI cannot act.These data confirm that the inclusion of two novel CTCF-based CIs of human origin completely abrogated the formation of tumors caused by enhancer-mediated activation of an oncogene in vivo.The ability of these two new insulator elements to block the crosstalk between powerful vector enhancers and cellular regulatory elements increase the safety of SIN LVs and justify their prompt adoption in future gene therapy applications

AB012. Transcriptional and chromatin profiling reveals the molecular architecture and druggable vulnerabilities of thymic epithelial tumors (TETs)

Thymic epithelial tumors (TETs) have been profiled to the present moment mainly through several analyses of FFPE samples. Despite the leap forward brought by the TCGA, several questions remain still unsolved. Among these, TETs are characterized by a strong component of immune infiltrate which makes the transcriptomic analyses conducted so far scarcely interpretable to profile stromal subpopulations constitutive of the tumor. Furthermore, rarely correspondent healthy tissue is available due to the lipomatous atrophy of aged thymi. Therefore, the recent report of (I) isolation, (II) propagation (III) and characterization of human thymic epithelial cells (TECs) and their capacity to reconstitute the functional organ ex vivo and in vivo, represents a novel approach to study the biology of both healthy and neoplastic thymi. Human thymic biopsies (both healthy and neoplastic) were digested and plated on a lethally irradiated murine feeder layer. Both RNA-Seq and CUTANDTAG were performed on cultivated TECs at different passages. Cultured TECs were injected with human thymic interstitial cells into rat decellularized scaffolds and cultivated for 10–12 days. sc-RNA Seq is currently being performed on both healthy and neoplastic thymic mini-organs and their correspondent primary tissues. Here show that we successfully cultivated a cohort of 21 clonogenic TECs in vitro including adult neoplastic TECs, their non-tumoral counterpart and pediatric TECs. We show that at the transcriptome level each class of TECs clusters independently and that neoplastic TECs belong to the same cloud independently from thymoma histotype. Around 1,400 differentially expressed genes (DEGs) can be found when comparing adult neoplastic and non-neoplastic counterpart, among which around 70 are transcription factors. Importantly, we prove for the first time that clonogenic TECs derived from TETs can repopulate a decellularized rat scaffold and recreate a 3D architecture mimicking the primary tumor. This work demonstrates that this culture system allows the expansion of clonogenic TECs from both tumor samples and their non-tumoral counterpart. Those cells, when transplanted into decellularized thymi, reproduce the architecture of the primary tissue, showing that TETs contain progenitor/stem epithelial cells. We are currently characterizing TECs at the transcriptomic and epigenomic level with aim of identifying new druggable targets prior to clinical trials

GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders

Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23 microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development and behavioral hallmarks remains largely unknown.We integrated proteomic and transcriptomic profiling of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23 dosage–dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis as a molecular pathway amenable to therapeutic intervention in ASD

KMT2B Is Selectively Required for Neuronal Transdifferentiation, and Its Loss Exposes Dystonia Candidate Genes

Transdifferentiation of fibroblasts into induced neuronal cells (iNs) by the neuron-specific transcription factors Brn2, Myt1l, and Ascl1 is a paradigmatic example of inter-lineage conversion across epigenetically distant cells. Despite tremendous progress regarding the transcriptional hierarchy underlying transdifferentiation, the enablers of the concomitant epigenome resetting remain to be elucidated. Here, we investigated the role of KMT2A and KMT2B, two histone H3 lysine 4 methylases with cardinal roles in development, through individual and combined inactivation. We found that Kmt2b, whose human homolog's mutations cause dystonia, is selectively required for iN conversion through suppression of the alternative myocyte program and induction of neuronal maturation genes. The identification of KMT2B-vulnerable targets allowed us, in turn, to expose, in a cohort of 225 patients, 45 unique variants in 39 KMT2B targets, which represent promising candidates to dissect the molecular bases of dystonia. Barbagiovanni et al. demonstrate that KMT2B, in contrast to KMT2A, is fundamental for the epigenetic and transcriptomic resetting underlying transdifferentiation of fibroblasts into induced neuronal cells (iNs), acting both in the suppression of alternative fates and in the promotion of iN maturation. Transdifferentiation-specific KMT2B targets reveal dystonia-causative gene candidates

From cohorts to molecules: Adverse impacts of endocrine disrupting mixtures

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay.ISSN:0036-8075ISSN:1095-920

From cohorts to molecules : Adverse impacts of endocrine disrupting mixtures

Convergent evidence associates exposure to endocrine disrupting chemicals (EDCs) with major human diseases, even at regulation-compliant concentrations. This might be because humans are exposed to EDC mixtures, whereas chemical regulation is based on a risk assessment of individual compounds. Here, we developed a mixture-centered risk assessment strategy that integrates epidemiological and experimental evidence. We identified that exposure to an EDC mixture in early pregnancy is associated with language delay in offspring. At human-relevant concentrations, this mixture disrupted hormone-regulated and disease-relevant regulatory networks in human brain organoids and in the model organisms Xenopus leavis and Danio rerio, as well as behavioral responses. Reinterrogating epidemiological data, we found that up to 54% of the children had prenatal exposures above experimentally derived levels of concern, reaching, for the upper decile compared with the lowest decile of exposure, a 3.3 times higher risk of language delay