Integrated molecular profiling of patient-derived ovarian cancer models identifies clinically relevant signatures and tumor vulnerabilities.

High-grade serous ovarian carcinoma (HGSOC) is a highly aggressive and intractable neoplasm, mainly because of its rapid dissemination into the abdominal cavity, a process that is favored by tumor-associated peritoneal ascites. The precise molecular alterations involved in HGSOC onset and progression remain largely unknown due to the high biological and genetic heterogeneity of this tumor. We established a set of different tumor samples (termed the As11-set) derived from a single HGSOC patient, consisting of peritoneal ascites, primary tumor cells, ovarian cancer stem cells (OCSC) and serially propagated tumor xenografts. The As11-set was subjected to an integrated RNA-seq and DNA-seq analysis which unveiled molecular alterations that marked the different types of samples. Our profiling strategy yielded a panel of signatures relevant in HGSOC and in OCSC biology. When such signatures were used to interrogate the TCGA dataset from HGSOC patients, they exhibited prognostic and predictive power. The molecular alterations also identified potential vulnerabilities associated with OCSC, which were then tested functionally in stemness-related assays. As a proof of concept, we defined PI3K signaling as a novel druggable target in OCSC.

Site-specific integration and tailoring of cassette design for sustainable gene transfer.

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

Tumor microenvironment-induced FOXM1 regulates ovarian cancer stemness.

In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

The first reported generation of several induced pluripotent stem cell lines from homozygous and heterozygous Huntington's disease patients demonstrates mutation related enhanced lysosomal activity.

Neuronal disorders, like Huntington's disease (HD), are difficult to study, due to limited cell accessibility, late onset manifestations, and low availability of material. The establishment of an in vitro model that recapitulates features of the disease may help understanding the cellular and molecular events that trigger disease manifestations. Here, we describe the generation and characterization of a series of induced pluripotent stem (iPS) cells derived from patients with HD, including two rare homozygous genotypes and one heterozygous genotype. We used lentiviral technology to transfer key genes for inducing reprogramming. To confirm pluripotency and differentiation of iPS cells, we used PCR amplification and immunocytochemistry to measure the expression of marker genes in embryoid bodies and neurons. We also analyzed teratomas that formed in iPS cell-injected mice. We found that the length of the pathological CAG repeat did not increase during reprogramming, after long term growth in vitro, and after differentiation into neurons. In addition, we observed no differences between normal and mutant genotypes in reprogramming, growth rate, caspase activation or neuronal differentiation. However, we observed a significant increase in lysosomal activity in HD-iPS cells compared to control iPS cells, both during self-renewal and in iPS-derived neurons. In conclusion, we have established stable HD-iPS cell lines that can be used for investigating disease mechanisms that underlie HD. The CAG stability and lysosomal activity represent novel observations in HD-iPS cells. In the future, these cells may provide the basis for a powerful platform for drug screening and target identification in HD.

Single cell-derived spheroids capture the self-renewing subpopulations of metastatic ovarian cancer.

High Grade Serous Ovarian cancer (HGSOC) is a major unmet need in oncology, due to its precocious dissemination and the lack of meaningful human models for the investigation of disease pathogenesis in a patient-specific manner. To overcome this roadblock, we present a new method to isolate and grow single cells directly from patients' metastatic ascites, establishing the conditions for propagating them as 3D cultures that we refer to as single cell-derived metastatic ovarian cancer spheroids (sMOCS). By single cell RNA sequencing (scRNAseq) we define the cellular composition of metastatic ascites and trace its propagation in 2D and 3D culture paradigms, finding that sMOCS retain and amplify key subpopulations from the original patients' samples and recapitulate features of the original metastasis that do not emerge from classical 2D culture, including retention of individual patients' specificities. By enabling the enrichment of uniquely informative cell subpopulations from HGSOC metastasis and the clonal interrogation of their diversity at the functional and molecular level, this method provides a powerful instrument for precision oncology in ovarian cancer.

A cell-of-origin epigenetic tracer reveals clinically distinct subtypes of high-grade serous ovarian cancer.

BACKGROUND: High-grade serous ovarian cancer (HGSOC) is a major unmet need in oncology. The remaining uncertainty on its originating tissue has hampered the discovery of molecular oncogenic pathways and the development of effective therapies. METHODS: We used an approach based on the retention in tumors of a DNA methylation trace (OriPrint) that distinguishes the two putative tissues of origin of HGSOC, the fimbrial (FI) and ovarian surface epithelia (OSE), to stratify HGSOC by several clustering methods, both linear and non-linear. The identified tumor subtypes (FI-like and OSE-like HGSOC) were investigated at the RNAseq level to stratify an in-house cohort of macrodissected HGSOC FFPE samples to derive overall and disease-free survival and identify specific transcriptional alterations of the two tumor subtypes, both by classical differential expression and weighted correlation network analysis. We translated our strategy to published datasets and verified the co-occurrence of previously described molecular classification of HGSOC. We performed cytokine analysis coupled to immune phenotyping to verify alterations in the immune compartment associated with HGSOC. We identified genes that are both differentially expressed and methylated in the two tumor subtypes, concentrating on PAX8 as a bona fide marker of FI-like HGSOC. RESULTS: We show that: - OriPrint is a robust DNA methylation tracer that exposes the tissue of origin of HGSOC. - The tissue of origin of HGSOC is the main determinant of DNA methylation variance in HGSOC. - The tissue of origin is a prognostic factor for HGSOC patients. - FI-like and OSE-like HGSOC are endowed with specific transcriptional alterations that impact patients' prognosis. - OSE-like tumors present a more invasive and immunomodulatory phenotype, compatible with its worse prognostic impact. - Among genes that are differentially expressed and regulated in FI-like and OSE-like HGSOC, PAX8 is a bona fide marker of FI-like tumors. CONCLUSIONS: Through an integrated approach, our work demonstrates that both FI and OSE are possible origins for human HGSOC, whose derived subtypes are both molecularly and clinically distinct. These results will help define a new roadmap towards rational, subtype-specific therapeutic inroads and improved patients' care.

Human Cortical Organoids Expose a Differential Function of GSK3 on Cortical Neurogenesis.

The regulation of the proliferation and polarity of neural progenitors is crucial for the development of the brain cortex. Animal studies have implicated glycogen synthase kinase 3 (GSK3) as a pivotal regulator of both proliferation and polarity, yet the functional relevance of its signaling for the unique features of human corticogenesis remains to be elucidated. We harnessed human cortical brain organoids to probe the longitudinal impact of GSK3 inhibition through multiple developmental stages. Chronic GSK3 inhibition increased the proliferation of neural progenitors and caused massive derangement of cortical tissue architecture. Single-cell transcriptome profiling revealed a direct impact on early neurogenesis and uncovered a selective role of GSK3 in the regulation of glutamatergic lineages and outer radial glia output. Our dissection of the GSK3-dependent transcriptional network in human corticogenesis underscores the robustness of the programs determining neuronal identity independent of tissue architecture.

HOXB7 overexpression in lung cancer is a hallmark of acquired stem-like phenotype.

HOXB7 is a homeodomain (HOX) transcription factor involved in regional body patterning of invertebrates and vertebrates. We previously identified HOXB7 within a ten-gene prognostic signature for lung adenocarcinoma, where increased expression of HOXB7 was associated with poor prognosis. This raises the question of how HOXB7 overexpression can influence the metastatic behavior of lung adenocarcinoma. Here, we analyzed publicly available microarray and RNA-seq lung cancer expression datasets and found that HOXB7-overexpressing tumors are enriched in gene signatures characterizing adult and embryonic stem cells (SC), and induced pluripotent stem cells (iPSC). Experimentally, we found that HOXB7 upregulates several canonical SC/iPSC markers and sustains the expansion of a subpopulation of cells with SC characteristics, through modulation of LIN28B, an emerging cancer gene and pluripotency factor, which we discovered to be a direct target of HOXB7. We validated this new circuit by showing that HOXB7 enhances reprogramming to iPSC with comparable efficiency to LIN28B or its target c-MYC, which is a canonical reprogramming factor.

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.