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BRCA1 and BRCA2 both function in DNA double-strand break repair by homologous recombination (HR). Due to their HR defect, BRCA1/2-deficient cancers are sensitive to poly(ADP-ribose) polymerase inhibitors (PARPis), but they eventually acquire resistance. Preclinical studies yielded several PARPi resistance mechanisms that do not involve BRCA1/2 reactivation, but their relevance in the clinic remains elusive. To investigate which BRCA1/2-independent mechanisms drive spontaneous resistance in vivo, we combine molecular profiling with functional analysis of HR of matched PARPi-naive and PARPi-resistant mouse mammary tumors harboring large intragenic deletions that prevent reactivation of BRCA1/2. We observe restoration of HR in 62% of PARPi-resistant BRCA1-deficient tumors but none in the PARPi-resistant BRCA2-deficient tumors. Moreover, we find that 53BP1 loss is the prevalent resistance mechanism in HR-proficient BRCA1-deficient tumors, whereas resistance in BRCA2-deficient tumors is mainly induced by PARG loss. Furthermore, combined multi-omics analysis identifies additional genes and pathways potentially involved in modulating PARPi response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Somatic hotspot mutations and structural amplifications and fusions that affect fibroblast growth factor receptor 2 (encoded by FGFR2) occur in multiple types of cancer 1 . However, clinical responses to FGFR inhibitors have remained variable 1-9 , emphasizing the need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. Here we apply transposon-based screening 10,11 and tumour modelling in mice 12,13 , and find that the truncation of exon 18 (E18) of Fgfr2 is a potent driver mutation. Human oncogenomic datasets revealed a diverse set of FGFR2 alterations, including rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations, each causing the transcription of E18-truncated FGFR2 (FGFR2 ΔE18 ). Functional in vitro and in vivo examination of a compendium of FGFR2 ΔE18 and full-length variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. By contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct landscape of cooperating driver genes. This suggests that genomic alterations that generate stable FGFR2 ΔE18 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumour models, and in a clinical trial. We propose that cancers containing any FGFR2 variant with a truncated E18 should be considered for FGFR-targeted therapies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

BRCA1-mutated breast cancer is primarily driven by DNA copy-number alterations (CNAs) containing large numbers of candidate driver genes. Validation of these candidates requires novel approaches for high-throughput in vivo perturbation of gene function. Here we develop genetically engineered mouse models (GEMMs) of BRCA1-deficient breast cancer that permit rapid introduction of putative drivers by either retargeting of GEMM-derived embryonic stem cells, lentivirus-mediated somatic overexpression or in situ CRISPR/Cas9-mediated gene disruption. We use these approaches to validate Myc, Met, Pten and Rb1 as bona fide drivers in BRCA1-associated mammary tumorigenesis. Iterative mouse modeling and comparative oncogenomics analysis show that MYC-overexpression strongly reshapes the CNA landscape of BRCA1-deficient mammary tumors and identify MCL1 as a collaborating driver in these tumors. Moreover, MCL1 inhibition potentiates the in vivo efficacy of PARP inhibition (PARPi), underscoring the therapeutic potential of this combination for treatment of BRCA1-mutated cancer patients with poor response to PARPi monotherapy.

Background: Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary., Results: We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations., Conclusions: XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR .

In human cancers, FGFR signaling is frequently hyperactivated by deregulation of FGF ligands or by activating mutations in the FGFR receptors such as gene amplifications, point mutations, and gene fusions. As such, FGFR inhibitors are considered an attractive therapeutic strategy for patients with mutations in FGFR family members. We previously identified Fgfr2 as a key driver of invasive lobular carcinoma (ILC) in an in vivo insertional mutagenesis screen using the Sleeping Beauty transposon system. Here we explore whether these FGFR-driven ILCs are sensitive to the FGFR inhibitor AZD4547 and use transposon mutagenesis in these tumors to identify potential mechanisms of resistance to therapy. Combined with RNA sequencing-based analyses of AZD4547-resistant tumors, our in vivo approach identified several known and novel potential resistance mechanisms to FGFR inhibition, most of which converged on reactivation of the canonical MAPK-ERK signaling cascade. Observed resistance mechanisms included mutations in the tyrosine kinase domain of FGFR2, overexpression of MET, inactivation of RASA1, and activation of the drug-efflux transporter ABCG2. ABCG2 and RASA1 were identified only from de novo transposon insertions acquired during AZD4547 treatment, demonstrating that insertional mutagenesis in mice is an effective tool for identifying potential mechanisms of resistance to targeted cancer therapies. Significance: These findings demonstrate that a combined approach of transcriptomics and insertional mutagenesis in vivo is an effective method for identifying potential targets to overcome resistance to therapy in the clinic. Cancer Res; 78(19); 5668-79. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Cancer is a complex disease in which cells progressively accumulate mutations disrupting their cellular processes. A fraction of these mutations drive tumourigenesis by affecting oncogenes or tumour suppressor genes, but many mutations are passengers with no clear contribution to tumour development. The advancement of DNA and RNA sequencing technologies has enabled in-depth analysis of thousands of human tumours from various tissues to perform systematic characterization of their (epi)genomes and transcriptomes in order to identify (epi)genetic changes associated with cancer. Combined with considerable progress in algorithmic development, this expansion in scale has resulted in the identification of many cancer-associated mutations, genes and pathways that are considered to be potential drivers of tumour development. However, it remains challenging to systematically identify drivers affected by complex genomic rearrangements and drivers residing in non-coding regions of the genome or in complex amplicons or deletions of copy-number driven tumours. Furthermore, functional characterization is challenging in the human context due to the lack of genetically tractable experimental model systems in which the effects of mutations can be studied in the context of their tumour microenvironment. In this respect, mouse models of human cancer provide unique opportunities for pinpointing novel driver genes and their detailed characterization. In this review, we provide an overview of approaches for complementing human studies with data from mouse models. We also discuss state-of-the-art technological developments for cancer gene discovery and validation in mice., (© 2018 The Authors.)

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Invasive lobular carcinoma (ILC) is the second most common breast cancer subtype and accounts for 8-14% of all cases. Although the majority of human ILCs are characterized by the functional loss of E-cadherin (encoded by CDH1), inactivation of Cdh1 does not predispose mice to develop mammary tumors, implying that mutations in additional genes are required for ILC formation in mice. To identify these genes, we performed an insertional mutagenesis screen using the Sleeping Beauty transposon system in mice with mammary-specific inactivation of Cdh1. These mice developed multiple independent mammary tumors of which the majority resembled human ILC in terms of morphology and gene expression. Recurrent and mutually exclusive transposon insertions were identified in Myh9, Ppp1r12a, Ppp1r12b and Trp53bp2, whose products have been implicated in the regulation of the actin cytoskeleton. Notably, MYH9, PPP1R12B and TP53BP2 were also frequently aberrated in human ILC, highlighting these genes as drivers of a novel oncogenic pathway underlying ILC development.

Insertional mutagenesis using engineered transposons is a potent forward genetic screening technique used to identify cancer genes in mouse model systems. In the analysis of these screens, transposon insertion sites are typically identified by targeted DNA-sequencing and subsequently assigned to predicted target genes using heuristics. As such, these approaches provide no direct evidence that insertions actually affect their predicted targets or how transcripts of these genes are affected. To address this, we developed IM-Fusion, an approach that identifies insertion sites from gene-transposon fusions in standard single- and paired-end RNA-sequencing data. We demonstrate IM-Fusion on two separate transposon screens of 123 mammary tumors and 20 B-cell acute lymphoblastic leukemias, respectively. We show that IM-Fusion accurately identifies transposon insertions and their true target genes. Furthermore, by combining the identified insertion sites with expression quantification, we show that we can determine the effect of a transposon insertion on its target gene(s) and prioritize insertions that have a significant effect on expression. We expect that IM-Fusion will significantly enhance the accuracy of cancer gene discovery in forward genetic screens and provide initial insight into the biological effects of insertions on candidate cancer genes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cells deficient in the Brca1 and Brca2 genes have reduced capacity to repair DNA double-strand breaks by homologous recombination and consequently are hypersensitive to DNA-damaging agents, including cisplatin and poly(ADP-ribose) polymerase (PARP) inhibitors. Here we show that loss of the MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells from DNA damage and rescues the lethality of Brca2-deficient embryonic stem cells. However, PTIP deficiency does not restore homologous recombination activity at double-strand breaks. Instead, its absence inhibits the recruitment of the MRE11 nuclease to stalled replication forks, which in turn protects nascent DNA strands from extensive degradation. More generally, acquisition of PARP inhibitors and cisplatin resistance is associated with replication fork protection in Brca2-deficient tumour cells that do not develop Brca2 reversion mutations. Disruption of multiple proteins, including PARP1 and CHD4, leads to the same end point of replication fork protection, highlighting the complexities by which tumour cells evade chemotherapeutic interventions and acquire drug resistance.

Cooperative behaviors are promoted by kin selection if the costs to the actor are smaller than the fitness benefits to the recipient, weighted by the coefficient of relatedness. In primates, cooperation occurs primarily among female dyads. Due to male dispersal before sexual maturity in many primate species, however, it is unknown whether there are sufficient opportunities for selective tolerance and occasional coalitionary support for kin selection to favor male nepotistic support. We studied the effect of the presence of male kin on correlates of male reproductive success (residence time, duration of high dominance rank) in non-natal male long-tailed macaques (Macaca fascicularis). We found that "related" (i.e., related at the half-sibling level or higher) males in a group have a significantly higher probability to remain in the non-natal group compared to males without relatives. Moreover, males stayed longer in a group when a relative was present at group entry or joined the same group within 3 months upon arrival. Males with co-residing relatives also maintained a high rank for longer than those without. To our knowledge, this is the first demonstration of a potential nepotistic effect on residence and rank maintenance among non-natal males in a social system without long-term alliances.

In two experiments and two different research paradigms, we tested the hypothesis that Zen meditation increases access to accessible but unconscious information. Zen practitioners who meditated in the lab performed better on the Remote Associate Test (RAT; Mednick, 1962) than Zen practitioners who did not meditate. In a new, second task, it was observed that Zen practitioners who meditated used subliminally primed words more than Zen practitioners who did not meditate. Practical and theoretical implications are discussed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Tool making or modification to produce a tool of apparent improved functionality has rarely been reported in monkeys, especially when tools are used outside the context of food acquisition. We report on an observation of selection, modification and use of splinters for hygiene purposes in a male mandrill. The zoo-housed animal was video-recorded breaking splinters in sequence to use them underneath his toenails. This record brings forward new evidence that the ability to use and modify tools is not limited to apes and some New World monkeys but is also apparent in Old Word monkeys., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In the recent past, application of DNA genotyping techniques has enabled researchers to more accurately test relationships between dominance rank (DR), mating success (MS) and reproductive success (RS). Paternity studies often reveal that reproductive outcome does not always correlate with male DR and/or MS and thus open room for discussion and interpretation of alternative reproductive tactics of both sexes. In this study, we analysed male DR, MS and RS in a group of bonobos at Twycross Zoo (UK). Genetic relationships were determined using 8 tetrameric microsatellite loci. Despite clear and asymmetric dominance relationships, analysed using normalised David's scores based on a dyadic index of dominance among the group's 3 mature males, we found that the most dominant male did not sire the most offspring. In fact, both infants conceived during the observation period were found to be sired by the lower-ranking males. Although the alpha male had almost exclusive mating access to one of the females during the time she was showing a maximal anogenital swelling, her infant was sired by the lowest-ranking male who mostly mated with her when outside the maximal swelling period. This result suggests that either sperm competition operates and/or ovulation is decoupled from the phase of maximal anogenital swelling which could allow greater female choice., (Copyright 2006 S. Karger AG, Basel.)

Genealogical relatedness is thought to be an important causal factor in the evolution of cooperation. We inferred relatedness on the basis of 11 blood protein markers using the Queller and Goodnight index of relatedness in a macaque population with long-term demographic records. This estimate reflected independently determined pedigree relationships in our data set. Mean relatedness among all members of a social group was 0.10 but much higher levels of relatedness (0.30-0.47) were found among the members of matrilineal families with a high or intermediate social rank. Groups of dispersing males that had been born into the same social group were sometimes closely related (0.43 and 0.58), but they could also be less related (0.08). We found that the pattern of distribution of relatedness was associated with gene flow and differential reproduction in males, rather than with group fission and the presence of geographical barriers.

Long-tailed macaques (Macaca fascicularis) live in social groups consisting of resident adult females and their offspring, and immigrant males. Subadult males leave their birth group, and might establish themselves as reproducing males in another group. Females do not leave their birth group. Such a social pattern might have consequences for the genetic differentiation between groups and the genetic relationships within groups. In a field study of long-tailed macaques (Macaca fascicularis) in Ketambe, Sumatra, Indonesia, blood samples were taken from individuals in seven adjacent social groups. Electrophoretic analysis showed 17 blood proteins and enzymes to be polymorphic, allowing the computation of heterozygosities and of the F-statistics. Of the F-statistics, F(IS) indicates the deviation from Hardy-Weinberg equilibrium averaged over local populations, FST indicates the differentiation in allele frequency between local populations, and F(IT) indicates the deviation from Hardy-Weinberg equilibrium over the total population. In a computer simulation of the population of long-tailed macaques using many loci with many neutral alleles, F(IS) and FST values proved to be characteristic for a certain demography and life history of the population, and proved not to depend upon the number of alleles or level of heterozygosity. FST values found in the simulation were compatible to those found in the field; in the simulation, values for F(IS) and F(IT) were consistently negative. The explanation for the negative F(IS) appears to be that genetic drift causes differentiation in allele frequencies between groups, and that due to this differentiation, allele frequencies differ between resident females and immigrant males, leading to offspring with an excess of heterozygotes (negative F(IS)) relative to the expectation based upon the overall allele frequency. The excess of heterozygotes might imply that slightly deleterious alleles are protected from selection. A population with a social structure and differential migration of the sexes is liable to accumulate deleterious recessives and, as a consequence, to be very sensitive to inbreeding on disruption of the social structure, as for instance in zoos.

Long-tailed macaques (Macaca fascicularis Raffles, 1821) were captured at various locations in the north of Sumatra as part of a study on social behaviour and genetic relationships. We used individual cage traps, a group trap, a blowpipe and an air-pressure rifle. Provided that the monkeys were willing to take bait, individual cage traps proved most successful; they gave a high capture rate with minimal disturbance of the group. Success with young juveniles and peripheral animals could be improved by placing elevated traps in the centre and in clusters at the periphery of the trapping site. Trapping had no clear lasting effect on the natural behaviour of the animals.