Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

FOXA1 repression drives lineage plasticity and immune heterogeneity in bladder cancers with squamous differentiation.

Cancers arising from the bladder urothelium often exhibit lineage plasticity with regions of urothelial carcinoma adjacent to or admixed with regions of divergent histomorphology, most commonly squamous differentiation. To define the biologic basis for and clinical significance of this morphologic heterogeneity, here we perform integrated genomic analyses of mixed histology bladder cancers with separable regions of urothelial and squamous differentiation. We find that squamous differentiation is a marker of intratumoral genomic and immunologic heterogeneity in patients with bladder cancer and a biomarker of intrinsic immunotherapy resistance. Phylogenetic analysis confirms that in all cases the urothelial and squamous regions are derived from a common shared precursor. Despite the presence of marked genomic heterogeneity between co-existent urothelial and squamous differentiated regions, no recurrent genomic alteration exclusive to the urothelial or squamous morphologies is identified. Rather, lineage plasticity in bladder cancers with squamous differentiation is associated with loss of expression of FOXA1, GATA3, and PPARG, transcription factors critical for maintenance of urothelial cell identity. Of clinical significance, lineage plasticity and PD-L1 expression is coordinately dysregulated via FOXA1, with patients exhibiting morphologic heterogeneity pre-treatment significantly less likely to respond to immune checkpoint inhibitors.

Bone Marrow Surveillance of Pediatric Cancer Survivors Identifies Clones that Predict Therapy-Related Leukemia.

PURPOSE: Therapy-related myelodysplastic syndrome and acute leukemias (t-MDS/AL) are a major cause of nonrelapse mortality among pediatric cancer survivors. Although the presence of clonal hematopoiesis (CH) in adult patients at cancer diagnosis has been implicated in t-MDS/AL, there is limited published literature describing t-MDS/AL development in children. EXPERIMENTAL DESIGN: We performed molecular characterization of 199 serial bone marrow samples from 52 patients treated for high-risk neuroblastoma, including 17 with t-MDS/AL (transformation), 14 with transient cytogenetic abnormalities (transient), and 21 without t-MDS/AL or cytogenetic alterations (neuroblastoma-treated control). We also evaluated for CH in a cohort of 657 pediatric patients with solid tumor. RESULTS: We detected at least one disease-defining alteration in all cases at t-MDS/AL diagnosis, most commonly TP53 mutations and KMT2A rearrangements, including involving two novel partner genes (PRDM10 and DDX6). Backtracking studies identified at least one t-MDS/AL-associated mutation in 13 of 17 patients at a median of 15 months before t-MDS/AL diagnosis (range, 1.3-32.4). In comparison, acquired mutations were infrequent in the transient and control groups (4/14 and 1/21, respectively). The relative risk for development of t-MDS/AL in the presence of an oncogenic mutation was 8.8 for transformation patients compared with transient. Unlike CH in adult oncology patients, TP53 mutations were only detectable after initiation of cancer therapy. Last, only 1% of pediatric patients with solid tumor evaluated had CH involving myeloid genes. CONCLUSIONS: These findings demonstrate the clinical relevance of identifying molecular abnormalities in predicting development of t-MDS/AL and should guide the formation of intervention protocols to prevent this complication in high-risk pediatric patients.

An autoimmune stem-like CD8 T cell population drives type 1 diabetes.

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, ß-cell-specific CD8 T cells destroy insulin-producing ß-cells. Here we followed the fate of ß-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy ß-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain ß-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.

Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.

Chromosome-autonomous feedback down-regulates meiotic DNA break competence upon synaptonemal complex formation.

The number of DNA double-strand breaks (DSBs) initiating meiotic recombination is elevated in Saccharomyces cerevisiae mutants that are globally defective in forming crossovers and synaptonemal complex (SC), a protein scaffold juxtaposing homologous chromosomes. These mutants thus appear to lack a negative feedback loop that inhibits DSB formation when homologs engage one another. This feedback is predicted to be chromosome autonomous, but this has not been tested. Moreover, what chromosomal process is recognized as "homolog engagement" remains unclear. To address these questions, we evaluated effects of homolog engagement defects restricted to small portions of the genome using karyotypically abnormal yeast strains with a homeologous chromosome V pair, monosomic V, or trisomy XV. We found that homolog engagement-defective chromosomes incurred more DSBs, concomitant with prolonged retention of the DSB-promoting protein Rec114, while the rest of the genome remained unaffected. SC-deficient, crossover-proficient mutants ecm11 and gmc2 experienced increased DSB numbers diagnostic of homolog engagement defects. These findings support the hypothesis that SC formation provokes DSB protein dissociation, leading in turn to loss of a DSB competent state. Our findings show that DSB number is regulated in a chromosome-autonomous fashion and provide insight into how homeostatic DSB controls respond to aneuploidy during meiosis.

Sequencing Spo11 Oligonucleotides for Mapping Meiotic DNA Double-Strand Breaks in Yeast.

Meiosis is a specialized form of cell division resulting in reproductive cells with a reduced, usually haploid, genome complement. A key step after premeiotic DNA replication is the occurrence of homologous recombination at multiple places throughout the genome, initiated with the formation of DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like protein Spo11. DSBs are distributed non-randomly in genomes, and understanding the mechanisms that shape this distribution is important for understanding how meiotic recombination influences heredity and genome evolution. Several methods exist for mapping where Spo11 acts. Of these, sequencing of Spo11-associated oligonucleotides (Spo11 oligos) is the most precise, specifying the locations of DNA breaks to the base pair. In this chapter we detail the steps involved in Spo11-oligo mapping in the SK1 strain of budding yeast Saccharomyces cerevisiae, from harvesting cells of highly synchronous meiotic cultures, through preparation of sequencing libraries, to the mapping pipeline used for processing the data.

Numerical and spatial patterning of yeast meiotic DNA breaks by Tel1.

The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Mechanisms of this regulation remain poorly understood. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to reveal aspects of the contribution of the Saccharomyces cerevisiae DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM). A tel1Δ mutant has globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tel1 mutation similarly increases Spo11-oligonucleotide levels but mutating known Tel1 phosphotargets on Hop1 and Rec114 does not, implicating Tel1 kinase activity and clarifying roles of Tel1 phosphorylation substrates. Deep sequencing of Spo11 oligonucleotides demonstrates that Tel1 shapes the genome-wide DSB landscape in unexpected ways. Early in meiosis, Tel1 absence causes widespread changes in DSB distributions across large chromosomal domains. Many of these changes are erased as meiosis proceeds, however, illustrating homeostatic behavior of DSB regulatory systems. We further find that effects of Tel1 are distinct but partially overlapping with previously described contributions of the recombination regulator Cst9 (also known as Zip3). Finally, we provide evidence indicating that Tel1-dependent DSB interference influences the population-average DSB landscape but also demonstrate that locally inhibitory effects of an artificial hotspot insertion can be both Tel1-independent and chromosomal context-dependent. Our findings delineate Tel1 roles in regulating number and location of DSBs and illuminate the complex interplay between Tel1 and other pathways for DSB control.

Phosphorylation of the Synaptonemal Complex Protein Zip1 Regulates the Crossover/Noncrossover Decision during Yeast Meiosis.

Interhomolog crossovers promote proper chromosome segregation during meiosis and are formed by the regulated repair of programmed double-strand breaks. This regulation requires components of the synaptonemal complex (SC), a proteinaceous structure formed between homologous chromosomes. In yeast, SC formation requires the "ZMM" genes, which encode a functionally diverse set of proteins, including the transverse filament protein, Zip1. In wild-type meiosis, Zmm proteins promote the biased resolution of recombination intermediates into crossovers that are distributed throughout the genome by interference. In contrast, noncrossovers are formed primarily through synthesis-dependent strand annealing mediated by the Sgs1 helicase. This work identifies a conserved region on the C terminus of Zip1 (called Zip1 4S), whose phosphorylation is required for the ZMM pathway of crossover formation. Zip1 4S phosphorylation is promoted both by double-strand breaks (DSBs) and the meiosis-specific kinase, MEK1/MRE4, demonstrating a role for MEK1 in the regulation of interhomolog crossover formation, as well as interhomolog bias. Failure to phosphorylate Zip1 4S results in meiotic prophase arrest, specifically in the absence of SGS1. This gain of function meiotic arrest phenotype is suppressed by spo11Δ, suggesting that it is due to unrepaired breaks triggering the meiotic recombination checkpoint. Epistasis experiments combining deletions of individual ZMM genes with sgs1-md zip1-4A indicate that Zip1 4S phosphorylation functions prior to the other ZMMs. These results suggest that phosphorylation of Zip1 at DSBs commits those breaks to repair via the ZMM pathway and provides a mechanism by which the crossover/noncrossover decision can be dynamically regulated during yeast meiosis.

Self-organization of meiotic recombination initiation: general principles and molecular pathways.

Recombination in meiosis is a fascinating case study for the coordination of chromosomal duplication, repair, and segregation with each other and with progression through a cell-division cycle. Meiotic recombination initiates with formation of developmentally programmed DNA double-strand breaks (DSBs) at many places across the genome. DSBs are important for successful meiosis but are also dangerous lesions that can mutate or kill, so cells ensure that DSBs are made only at the right times, places, and amounts. This review examines the complex web of pathways that accomplish this control. We explore how chromosome breakage is integrated with meiotic progression and how feedback mechanisms spatially pattern DSB formation and make it homeostatic, robust, and error correcting. Common regulatory themes recur in different organisms or in different contexts in the same organism. We review this evolutionary and mechanistic conservation but also highlight where control modules have diverged. The framework that emerges helps explain how meiotic chromosomes behave as a self-organizing system.

Homologue engagement controls meiotic DNA break number and distribution.

Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. Here we test the hypothesis that DSB control involves a network of intersecting negative regulatory circuits. Using multiple complementary methods, we show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. ZMM-dependent DSB control is genetically distinct from a pathway tying break formation to meiotic progression through the Ndt80 transcription factor. These counterintuitive findings suggest that homologous chromosomes that have successfully engaged one another stop making breaks. Genome-wide DSB maps uncover distinct responses by different subchromosomal domains to the ZMM mutation zip3 (also known as cst9), and show that Zip3 is required for the previously unexplained tendency of DSB density to vary with chromosome size. Thus, feedback tied to ZMM function contributes in unexpected ways to spatial patterning of recombination.

Site-specific cross-linking of TBP in vivo and in vitro reveals a direct functional interaction with the SAGA subunit Spt3.

The TATA-binding protein (TBP) is critical for transcription by all three nuclear RNA polymerases. In order to identify factors that interact with TBP, the nonnatural photoreactive amino acid rho-benzoyl-phenylalanine (BPA) was substituted onto the surface of Saccharomyces cerevisiae TBP in vivo. Cross-linking of these TBP derivatives in isolated transcription preinitiation complexes or in living cells reveals physical interactions of TBP with transcriptional coregulator subunits and with the general transcription factor TFIIA. Importantly, the results show a direct interaction between TBP and the SAGA coactivator subunits Spt3 and Spt8. Mutations on the Spt3-interacting surface of TBP significantly reduce the interaction of TBP with SAGA, show a corresponding decrease in transcription activation, and fail to recruit TBP to a SAGA-dependent promoter, demonstrating that the direct interaction of these factors is important for activated transcription. These results prove a key prediction of the model for stimulation of transcription at SAGA-dependent genes via Spt3. Our cross-linking data also significantly extend the known surfaces of TBP that directly interact with the transcriptional regulator Mot1 and the general transcription factor TFIIA.

Fine mapping of a major locus on chromosome 10 for exploratory and fear-like behavior in mice.

Advanced intercross lines (AIL) and interval-specific congenic strains (ISCS) were used to fine map previously coarsely defined quantitative trait loci (QTL) on Chromosomes 1, 10, and 19, influencing behaviors in the open Field (OF) and light-dark (LD) paradigms in mice. F12(A x B) AIL mice (N = 1130) were phenotyped, genotyped, and mapped. The ISCS were studied only in the telomeric Chromosome 10 region of interest, containing the exploratory and excitability QTL1 (Exq1). The Chromosome 10 Exq1 and Chromosome 19 Exq4 loci mapped robustly in the AIL. The most significant QTL findings (2.0 LOD score intervals; peak; LOD score) came from the TD15 and LD transitions traits, yielding estimated intervals of 2.2 cM for Exq1 (71.3-73.5 cM; peak 72.3 cM; LOD 11.9) and 9.0 cM for Exq4 (29.0-38.2 cM; peak 34 cM; LOD 4.2). The replicated QTLs on Chromosome 1 failed to map in this AIL population. The ISCS data confirmed Exq1 loci in general. However, the ISCS data were complex and less definitive for localizing the Exq1 loci. These exploratory and fear-like behaviors result from inheriting "many small things," namely, QTL explaining 2%-7% of the phenotypic variance. These results highlight the challenges of positionally cloning loci of small effect for complex traits. In particular, fine-mapping success may depend on the genetic architecture underlying complex traits.

Function of a eukaryotic transcription activator during the transcription cycle.

Site-specific photocrosslinkers positioned within the central transcription-activating region of yeast Gcn4 were used to identify, in an unbiased way, three polypeptides in direct physical proximity to the activator during the process of transcription activation. Crosslinking was specific and did not change during different steps of the transcription cycle. The crosslinking targets were identified as Tra1, Gal11, and Taf12, subunits of four complexes (SAGA, NuA4, Mediator, and TFIID) known to play a role in gene regulation. Using this crosslinking assay, an activating region mutant, and extracts depleted of individual complexes containing the crosslinking targets, we found that contact with Tra1/SAGA is critical for activation, Gal11 contact has a modest effect on activation, and contact with TFIID and NuA4 is of little or no importance for activation under our conditions. Thus, a single activating region contacts multiple factors, and each contact makes differential contributions to transcriptional activation.