Biology subjects

Schimenti, J. C.

Publications and source records attributed to Schimenti, J. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Modeling of human MLH1 and MLH3 variants in mice reveals novel functions of MutLγ in meiotic DNA repair and the ovarian reserve

The mismatch repair proteins MLH1 and MLH3 form the MutL{gamma} complex that is essential for >90% of meiotic crossovers (CO) in mice. Although deletion of either gene causes infertility, we previously found that mouse models of certain human MLH1/3 missense variants caused elevated aneuploidy and progressive loss of female fecundity despite having a normal ovarian reserve. Here, we report that additional missense and null alleles caused significant pre-pubertal follicle loss independent of alterations to meiotic CO levels. Deletion of the DNA damage checkpoint kinase Chek2 (Chk2) partially rescued the oocyte pool of Mlh1- and Mlh3-deficient mice, indicating that defects in meiotic prophase I triggered oocyte elimination. In spermatocytes, both missense and null alleles caused abnormal {gamma}H2AX flares on autosomes during late pachynema and diplonema, extending into chromatin loops from the synaptonemal complex. These flares frequently contained unusually large foci of the ssDNA binding protein RPA as well as DNA end processing factors including CtIP, RAD50 and BLM, consistent with abnormal and extensive DNA resection. The results reveal a previously unrecognized meiotic function of MutL{gamma} in preventing DNA hyper-resection of spontaneous DNA breaks or of persistent recombination intermediates in late prophase I. Together with our previous studies, the results show that MLH1/3 variants can impair fertility through at least three mechanistically distinct routes: severe crossover loss sufficient to activate the metaphase I spindle assembly checkpoint; subthreshold crossover defects that increase aneuploidy and embryo loss; and abnormal late-prophase DNA processing that activates a CHEK2-dependent oocyte quality-control pathway and depletes the ovarian reserve, causing primary ovarian insufficiency (POI). Thus, MutL{gamma} has broader roles in maintaining meiotic genome integrity than can be explained by crossover formation alone.

genetics↗

bioRxiv2025

Embryonic Stem Cell-Specific Responses to DNA Replication Stress

Genome maintenance is of the utmost importance in stem cells, as mutations can be propagated and cause defects in derivative tissues. Many stem cell types display low mutation rates, with embryonic stem cells (ESCs) being a notable example. The bases for this property are unclear but may be achieved by optimization of various processes including high-fidelity DNA repair, cell cycle checkpoint controls, and hypersensitivity to genotoxic insults that trigger cell death. Here, we investigate the mechanisms underlying the unique responses of mouse ESCs (mESCs) to replication stress (RS) using an array of small molecule inhibitors and genotoxins. We find that whereas mESCs survive under acute RS in an ATR- and CHK1-dependent manner similar to somatic cells, they lack a strong G2/M checkpoint and fail to repair DNA crosslinks in the absence of ATR signaling. Despite the lack of a strong G2/M checkpoint, mESCs maintain a spindle assembly checkpoint (SAC). We posit that mESCs preferentially repair DNA crosslinks in S phase via homology-directed mechanisms, and cells that fail to complete repair before mitosis undergo mitotic catastrophe and cell death. These findings shed light on mutation avoidance mechanisms in ESCs that may extend to other stem cell types.

cell biology↗

bioRxiv2025

Gene combinations driving transformation of tubal epithelial organoids and differential sensitivity to high-grade serous ovarian carcinoma drugs

High-grade serous ovarian carcinoma (HGSC) is the sixth leading cause of cancer-related death among women. Many cases arise from the Fallopian tubal epithelium (TE), exhibit numerous mutations, and present heterogenous pathological features. However, the contribution of specific mutation combinations to cellular transformation, pathological phenotype and chemotherapeutic response remains poorly understood. Here, we used a Trp53-deficient mouse TE-derived organoid platform to perform combinatorial CRISPR mutagenesis of 20 candidate HGSC driver genes. Mutations in Nf1, Cdkn2a and Map2k4 were most frequently observed in transformed organoids. Upon transplantation into mice, those containing Map2k4 mutations predominantly gave rise to papillary-glandular histology, whereas those containing Nf1 mutations formed more mesenchymal-like carcinomas. Transcriptomic analysis revealed that Nf1-mutant tumors of all pathological phenotypes overexpressed the long non-coding RNA Pvt1, a marker associated with poor prognosis in HGSC patients. Map2k4-mutant organoids were more sensitive to paclitaxel and niraparib, while Nf1-mutant combinations responded better to trametinib. Notably, the removal of Rho kinase inhibitor (ROCKi) reduced trametinib sensitivity in both Map2k4- and Nf1-mutant organoids, underscoring the importance of culture conditions and potential antagonistic drug interactions in organoid-based drug screens. Collectively, our results demonstrate that TE-derived organoids coupled with combinatorial CRISPR mutagenesis provide a powerful system to unravel the genetic and phenotypic complexity of HGSC. In particular, we found that Map2k4 functions as a tumor suppressor that shapes distinct tumor histology and chemosensitivity, suggesting it as a potential therapeutic target in select HGSC cases.

cancer biology↗

bioRxiv2025

Chronic replication stress-mediated genomic instability disrupts placenta development in mice

Abnormal placentation drives many pregnancy-related pathologies and poor fetal outcomes, but the underlying molecular causes are understudied. Here, we show that persistent replication stress due to mutations in the MCM2-7 replicative helicase disrupts placentation and reduces embryo viability in mice. MCM-deficient embryos exhibited normal morphology but their placentae had a drastically diminished junctional zone (JZ). Whereas cell proliferation in the labyrinth zone (LZ) remained unaffected, JZ cell proliferation was reduced during development. MCM2-7 deficient trophoblast stem cells (TSCs) failed to maintain stemness, suggesting that replication stress affects the initial trophoblast progenitor pool in a manner that preferentially impacts the developing JZ. In contrast, pluripotency of mouse embryonic stem cells with MCM2-7 deficiency were not affected. Developing female mice deficient for FANCM, a protein involved in replication-associated DNA repair, also had placentae with a diminished JZ. These findings indicate that replication stress-induced genomic instability compromises embryo outcomes by impairing placentation.

developmental biology↗

bioRxiv2024

A high throughput CRISPR perturbation screen identifies epigenetic regulators impacting primordial germ cell development

Withdrawal StatementThe authors have withdrawn this manuscript due this manuscript merging with another preprint (BIORXIV/2024/580543). Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The merged preprint can be found at doi: 10.1101/2024.02.15.580543" The original version of 580543 described the development and characterization of the engineered mouse embryonic stem cells (ESCs) that could be differentiated into primordial germ cell-like cells (PGCLCs) in a scalable and efficient manner. Those cells were used to conduct the large screen (and other experiments) described in this withdrawn preprint. We merged the two manuscripts at the recommendation of a journal editor to which we sent the two papers for concurrent consideration. There are no substantive differences in the content merged preprint vs. this withdrawn preprint or the original version of 580543.

developmental biology↗

bioRxiv2024

Scalable and Efficient Generation of Mouse Primordial Germ Cell-like Cells

Primordial germ cells (PGCs) are the founder cells of the germline. The ability to generate PGC-like cells (PGCLCs) from pluripotent stem cells has advanced our knowledge of gametogenesis and holds promise for developing infertility treatments. However, generating an ample supply of PGCLCs for demanding applications such as large-scale genetic screens has been a limitation. Here, we demonstrated that simultaneous overexpression of 4 transcriptional factors - Nanog and three PGC master regulators Prdm1, Prdm14 and Tfap2c - in suspended mouse epiblast like cells (EpiLCs) and formative embryonic stem cells (ESCs) results in efficient and cost-effective production of PGCLCs. Nanog overexpression enhanced the PGC regulatory network, suppressed differentiation of somatic lineages, and maintained PGC fate. PGCLCs generated in this manner have transcriptomes similar to in vivo PGCs and are more advanced than cytokine-induced PGCLCs. These differentiated PGCLCs could be sustained over prolonged periods of culture and could differentiate into spermatogonia-like cells in vitro. We exploited this platform to conduct a bulk epigenome-wide CRISPRi screen to identify genes that decreased the efficiency of PGCLC formation when downregulated. One of the identified candidates was Ncor2, a transcriptional repressor that acts via recruitment of Class I and Class IIa histone deacetylases (HDACs). Consistently with this finding, the HDAC inhibitors valproic acid (VPA; an anti-convulsant) and sodium butyrate (SB; a widely-used dietary supplement) suppressed PGCLC differentiation. Furthermore, exposure of developing mouse embryos to SB or VPA caused hypospermatogenesis. This work demonstrates the feasibility of this platform to efficiently conduct large-scale functional screens of genes, chemicals, or other agents that may impact germline development.

cell biology↗

bioRxiv2023

Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

The distal region of the uterine (Fallopian) tube is commonly associated with high-grade serous carcinoma (HGSC), the predominant and most aggressive form of ovarian or extra-uterine cancer. Specific cell states and lineage dynamics of the adult tubal epithelium (TE) remain insufficiently understood, hindering efforts to determine the cell of origin for HGSC. Here, we report a comprehensive census of cell types and states of the mouse uterine tube. We show that distal TE cells expressing the stem/progenitor cell marker Slc1a3 can differentiate into both secretory (Ovgp1+) and ciliated (Fam183b+) cells. Inactivation of Trp53 and Rb1, whose pathways are commonly altered in HGSC, leads to elimination of targeted Slc1a3+ cells by apoptosis, thereby preventing their malignant transformation. In contrast, pre-ciliated cells (Krt5+, Prom1+, Trp73+) remain cancer-prone and give rise to serous tubal intraepithelial carcinomas and overt HGSC. These findings identify transitional pre-ciliated cells as a previously unrecognized cancer-prone cell state and point to pre-ciliation mechanisms as novel diagnostic and therapeutic targets.

cancer biology↗

bioRxiv2022

Integrative genome-scale analyses reveal post-transcriptional signatures of early human smallintestinal development in a directed differentiation organoid model

MicroRNAs (miRNAs) are important post-transcriptional gene regulators in organ development. To explore candidate roles for miRNAs in prenatal SI lineage specification in humans, we used a multi-omic analysis strategy in a directed differentiation model that programs human pluripotent stem cells toward the SI lineage. We leveraged small RNA-seq to define the changing miRNA landscape, and integrated chromatin run-on sequencing (ChRO-seq) and RNA-seq to define genes subject to significant post-transcriptional regulation across the different stages of differentiation. Our analyses showed that the elevation of miR-182 and reduction of miR-375 are key events during SI lineage specification. We demonstrated that loss of miR-182 leads to an increase in the foregut marker SOX2. We also used single-cell analyses in murine adult intestinal crypts to support a life-long role for miR-375 in the regulation of Zfp36l2. Finally, we uncovered opposing roles of SMAD4 and WNT signaling in regulating miR-375 expression during SI lineage specification. Beyond the mechanisms highlighted in this study, we also present a web-based application for exploration of post-transcriptional regulation and miRNA-mediated control in the context of early human SI development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/499825v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1ebf48eorg.highwire.dtl.DTLVardef@4a0d9org.highwire.dtl.DTLVardef@1dc3be9org.highwire.dtl.DTLVardef@1e14207_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2022

Imaging optically thick tissues simply and reproducibly: a practical guide to Lightsheet Macroscopy

Lightsheet microscopy offers an ideal method for imaging of large (mm-cm scale) biological tissues rendered transparent via optical clearing protocols. However the diversity of clearing technologies and tissue types, and how these are adapted to the microscope can make tissue mounting glitchy and somewhat irreproducible. Tissue preparation for imaging can involve glues and or equilibration in a variety of expensive and/or proprietary formulations. Here we present practical advice for mounting and capping cleared tissues in optical cuvettes for macroscopic imaging, providing a standardized 3D cell that can be imaged routinely and relatively inexpensively. We show that acrylic cuvettes should be non-aberrating with objective numerical apertures less than 0.65, and present an inexpensive tool for alignment and calibration of standard lightsheet parameters. Mouse embryo, liver and heart imaging are demonstrated as examples with practical recommendations for acquisition and post-processing.

bioengineering↗

bioRxiv2021

Sequencing Micronuclei Reveals the Landscape of Chromosomal Instability

Genome instability (GIN) is a main contributing factor to congenital and somatic diseases, but its sporadic occurrence in individual cell cycles makes it difficult to study mechanistically. One profound manifestation of GIN is the formation of micronuclei (MN), the engulfment of chromosomes or chromosome fragments in their own nuclear structures separate from the main nucleus. Here, we developed MN-seq, an approach for sequencing the DNA contained within micronuclei. We applied MN-seq to mice with mutations in Mcm4 and Rad9a, which disrupt DNA replication, repair, and damage responses. Data analysis and simulations show that centromere presence, fragment length, and a heterogenous landscape of chromosomal fragility all contribute to the patterns of DNA present within MN. In particular, we show that long genes, but also gene-poor regions, are associated with chromosome breaks that lead to the enrichment of particular genomic sequences in MN, in a genetic background-specific manner. Finally, we introduce single-cell micronucleus sequencing (scMN-seq), an approach to sequence the DNA present in MN of individual cells. Together, sequencing micronuclei provides a systematic approach for studying GIN and reveals novel molecular associations with chromosome breakage and segregation.

genomics↗

bioRxiv2021

In vivo versus in silico assessment of potentially pathogenic missense variants in human reproductive genes

Infertility is a heterogeneous condition, with genetic causes estimated to be involved in approximately half of the cases. High-throughput sequencing (HTS) is becoming an increasingly important tool for genetic diagnosis of diseases including idiopathic infertility, however, most rare or minor alleles revealed by HTS are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of population polymorphisms in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXOI, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction in the yeast 2 hybrid assay. Even though these genes are known to be essential for normal meiosis or spermiogenesis in mice, only one of the tested human variants (rs1460351219, encoding p.R581H in MCM9), which was observed in a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely-used algorithms to: 1) those present in ClinVar, and 2) those which have been evaluated in mice. We found that all the algorithms performed poorly in terms of predicting the effects of human missense variants that have been modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms, and emphasize the need for alternative and efficient in vitro or vivo functional validation models for more effective and accurate VUS delineation to either pathogenic or benign categories. SignificanceAlthough infertility is a substantial medical problem that affects up to 15% of couples, the potential genetic causes of idiopathic infertility have been difficult to decipher. This problem is complicated by the large number of genes that can cause infertility when perturbed, coupled with the large number of VUS that are present in the genomes of affected patients. Here, we present and analyze mouse modeling data of missense variants that are classified as deleterious by commonly-used pathogenicity prediction algorithms but which caused no detectible phenotype when introduced into mice by genome editing. We find that augmenting pathogenicity predictions with preliminary screens for biochemical defects substantially enhanced the proportion of prioritized variants that caused phenotypes in mice. The results emphasize that, in the absence of substantial improvements of in silico prediction tools or other compelling pre-existing evidence, in vivo analysis is crucial for confident attribution of infertility alleles.

genetics↗

bioRxiv2021

MLH1/3 variants causing aneuploidy, pregnancy loss, and premature reproductive aging

Most spontaneous pregnancy losses are a result of embryonic aneuploidy stemming from mis-segregation of chromosomes during meiosis. Proper disjunction of homologous chromosomes is dependent upon precise control of crossing-over, a process requiring the mismatch repair (MMR) genes MLH1 and MLH3. Both are required for fertility and completion of meiosis in mice. People inheriting variants in these genes are often at high risk for colorectal cancer and Lynch syndrome, yet the potential impacts of variants upon reproduction are unclear. To determine if MLH1/3 variants (namely single nucleotide polymorphisms, or SNPs) in human populations can cause reproductive abnormalities, we used a combination of computational predictions, yeast two-hybrid assays, and assays of MMR and recombination in yeast to select nine MLH1 and MLH3 variants for modeling in mice via genome editing. We identified 7 alleles that caused reproductive defects in mice including subfertility in females, male infertility, reduced sperm counts, and increased spermatocyte apoptosis. Remarkably, these alleles in females caused age-dependent decreases in litter size, and increased resorption of embryos during pregnancy. These outcomes were likely a consequence of reduced meiotic chiasmata, in turn causing an increase in misaligned chromosomes and univalents in meiotic metaphase I (MI). Our data indicate that segregating hypomorphic alleles of meiotic recombination genes in populations can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.

genetics↗

bioRxiv2020

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in [~]90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

genetics↗

bioRxiv2020

Single Cell Analysis Reveals Multi-faceted miR-375 Regulation of the Intestinal Crypt

The role of individual miRNAs in small intestinal (SI) epithelial homeostasis is under-explored. In this study, we discovered that miR-375 is among the most enriched miRNAs in intestinal crypts and stem cells (ISCs), especially facultative ISCs. We then showed by multiple manipulations, including CRISPR/Cas9 editing, that miR-375 is strongly suppressed by Wnt-signaling. Single-cell RNA-seq analysis of SI crypt-enriched cells from miR-375 knockout (375-KO) mice revealed elevated numbers of tuft cells and increased expression of pro-proliferative genes in ISCs. Accordingly, the genetic loss of miR-375 promoted resistance to helminth infection and enhanced the regenerative response to irradiation. The conserved effects of miR-375 were confirmed by gain-of-function studies in Drosophila midgut stem cells in vivo. Moreover, functional experiments in enteroids uncovered a regulatory relationship between miR-375 and Yap1 that controls cell survival. Finally, analysis of mouse model and clinical data revealed an inverse association between miR-375 levels and intestinal tumor development. HighlightsO_LImiR-375 is one of the most enriched miRNAs in ISCs, especially facultative ISCs. C_LIO_LImiR-375 modifies tuft cell abundance and pro-proliferative gene expression in ISCs. C_LIO_LILoss of miR-375 in mice enhances the host response to helminth infection and crypt regeneration. C_LIO_LIMouse and human intestinal cancer are associated with reduced miR-375 expression. C_LI eTOC BlurbSethupathy and colleagues show that miR-375 is a Wnt-responsive, ISC-enriched miRNA that serves as a break on intestinal crypt proliferation. They also show that miR-375 modulates tuft cell abundance and pro-proliferative gene expression in ISCs, that miR-375 loss enhances the host response to helminth infection as well as crypt regeneration post-irradiation, and its reduced expression is associated with intestinal cancer.

molecular biology↗

bioRxiv2020

Variants in RABL2A causing male infertility and ciliopathy

PurposeApproximately 7% of men suffer from infertility worldwide and sperm abnormalities are the major cause. Though genetic defects are thought to underlie a substantial fraction of all male infertility cases, the actual molecular bases are usually undetermined. Because the consequences of most genetic variants in populations are unknown, this complicates genetic diagnosis even after genome sequencing of patients. Some patients with ciliopathies, including primary ciliary dyskinesia (PCD) and Bardet-Biedl syndrome (BBS), also suffer from infertility because sperm flagella, which share several characteristics with cilia, are also affected in these patients. MethodsTo identify infertility-causing genetic variants in human populations, we used in silico predictions to identify potentially deleterious SNP (single nucleotide polymorphism) alleles of RABL2A, a gene essential for normal cilia and flagella function. Candidate variants were assayed for protein stability in vitro, and the destabilizing variants were modeled in mice using CRISPR/Cas9-mediated genome editing. The resulting mice were characterized phenotypically for reproductive and developmental defects. ResultsTwo of the SNP alleles, Rabl2L119F (rs80006029) and Rabl2V158F (rs200121688), destabilized the protein. Mice bearing these alleles exhibited PCD- and BBS-associated disorders including male infertility, early growth retardation, excessive weight gain in adulthood, heterotaxia, pre-axial polydactyly, neural tube defects (NTD) and hydrocephalus. ConclusionOur study identified and validated pathogenicity of two variants causing ciliopathies and male infertility in human populations, and identified phenotypes not previously described for null alleles of Rabl2.

genetics↗

bioRxiv2020

Sexually Dimorphic DNA Damage Responses and Mutation Avoidance in the Mouse Germline

Germ cells specified during fetal development form the foundation of the mammalian germline. These primordial germ cells (PGCs) undergo rapid proliferation, yet the germline is highly refractory to mutation accumulation compared to somatic cells. Importantly, while the presence of endogenous or exogenous DNA damage has the potential to impact PGCs, there is little known about how these cells respond to stressors. To better understand the DNA damage response (DDR) in these cells, we exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR in PGCs. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Additionally, the response to IR-induced DNA damage differs between female and male PGCs post-sex determination. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon de-repression. We also used whole genome single-cell DNA sequencing to reveal that genetic rescue of DNA repair-deficient germ cells (Fancm-/-) leads to increased mutation incidence and biases. Importantly, our work uncovers novel insights into how PGCs exposed to DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.

genetics↗

bioRxiv2020

A Reporter Mouse for In Vivo Detection of DNA Damage in Embryonic Germ Cells

Maintaining genome integrity in the germline is essential for survival and propagation of a species. In both mouse and human, germ cells originate during fetal development and are hypersensitive to both endogenous and exogenous DNA damaging agents. Currently, mechanistic understanding of how primordial germ cells respond to DNA damage is limited in part by the tools available to study these cells. We developed a mouse transgenic reporter strain expressing a 53BP1-mCherry fusion protein under the control of the Oct4{Delta}PE embryonic germ cell-specific promoter. This reporter binds sites of DNA double strand breaks (DSBs) on chromatin, forming foci. Using ionizing radiation as a DNA double strand break-inducing agent, we show that the transgenic reporter expresses specifically in the embryonic germ cells of both sexes and forms DNA damage induced foci in both a dose- and time-dependent manner. The dynamic time-sensitive and dose-sensitive DNA damage detection ability of this transgenic reporter, in combination with its specific expression in embryonic germ cells, makes it a versatile and valuable tool for increasing our understanding of DNA damage responses in these unique cells.

cell biology↗

bioRxiv2020

Most commonly mutated genes in High Grade Serous Ovarian Carcinoma are nonessential for ovarian surface epithelial stem cell transformation

High grade serous ovarian carcinoma (HGSOC) is the most lethal gynecological cancer and the 5th leading cause of cancer-related deaths of women in the USA. Disease-associated mutations have been identified by the Cancer Genome Atlas Research Network. However, aside from mutations in TP53 or alterations in the RB1 pathway that are extremely common in HGSOC, the contributions of other mutation combinations have been difficult to assess experimentally or with genomic data alone. Previous research identified ALDH+ stem cells of the ovarian surface epithelium (OSE) as one of the putative cells of HGSOC origin. Here, we performed combinatorial CRISPR mutagenesis of 20 putative HGSOC driver genes to identify mutation combinations that transformed OSE stem cells (OSE-SC) and non-stem cells (OSE-NS). Overrepresented mutations and mutation combinations were identified in all transformants and were investigated directly in targeted assays. Our results support the OSE stem cell theory of HGSOC initiation and suggest that most commonly mutated genes in HGSOC have no effect on OSE-SC transformation initiation. We suggest a model in which combined disruption of RB1 and PTEN, in addition to TP53 deficiency, constitutes a core set of mutations required for efficient transformation in vitro. A few previously uncharacterized mutation combinations further enhanced transformation but may have done so via TP53-related mechanisms. Together, our results identify mutation combinations that are critical for OSE-SC transformation and may contribute to more accurate modeling of HGSOC development. Our cancer driver screening methodology may also serve as a model for high throughput functional assessment of commonly mutated genes uncovered in other cancers by large scale sequencing.

cancer biology↗