Aneuploid Genomes Research Articles

Generating CRISPR-edited clonal lines of cultured Drosophila S2 cells.

CRISPR/Cas9 genome editing is a pervasive research tool due to its relative ease of use. However, some systems are not amenable to generating edited clones due to genomic complexity and/or difficulty in establishing clonal lines. For example, Drosophila Schneider 2 (S2) cells possess a segmental aneuploid genome and are challenging to single-cell select. Here, we describe a streamlined CRISPR/Cas9 methodology for knock-in and knock-out experiments in S2 cells, whereby an antibiotic resistance gene is inserted in-frame with the coding region of a gene-of-interest. By using selectable markers, we have improved the ease and efficiency for the positive selection of null cells using antibiotic selection in feeder layers followed by cell expansion to generate clonal lines. Using this method, we generated the first acentrosomal S2 cell lines by knocking-out centriole genes Polo-like Kinase 4/Plk4 or Ana2 as proof of concept. These strategies for generating gene-edited clonal lines will add to the collection of CRISPR tools available for cultured Drosophila cells by making CRISPR more practical and therefore improving gene function studies.

Chromosome-Level Assembly Reveals a Fifteen-Chromosome Aneuploid Genome and Environmental Adaptation Strategy of Chinese Traditional Medical Fungus Wolfiporia hoelen.

The sclerotia of Wolfiporia hoelen are one of the most important traditional Chinese medicines and foods commonly used in China, Japan, Korea, and other Asian countries. To provide a high-quality reference genome and deepen our understanding of the genome of W. hoelen to elucidate various biological phenomena. In this study, we assembled three genomes of W. hoelen using a combination of Nanopore and Illumina sequencing strategies. The fifteen-chromosome genome L7 of W. hoelen was assembled with two-sided telomere and rDNA sequences for the first time. The chromosome count was subsequently confirmed through collinearity analysis, correcting the previous belief that W. hoelen had only fourteen chromosomes. Moreover, the aneuploid genome was discovered in W. hoelen for the first time through sequencing depth analysis of different chromosomes, and only some strains of W. hoelen exhibit aneuploid genomes. According to the genome analysis of homokaryotic offspring and protoplast-isolated strains, a potential variation in chromosome allocation patterns was revealed. Moreover, the gene function enrichment analysis of genes on reduplicated chromosomes demonstrated that aneuploidy in the genome may be the result of environmental adaptation for W. hoelen. The discovery of an aneuploid genome also provides new ideas for genetic improvement of W. hoelen.

Genome size and chromosome number variation in sugarcane (Saccharum spp.) families and phenotypically contrasting Saccharum genotypes

AbstractModern sugarcane (Saccharum spp.) cultivars originated from an intricate hybridization process, resulting in complex polyploid and aneuploid genomes. Flow cytometry (FCM), which has emerged as a precise and fast method for research on genome size in plants, has potential to unveil key aspects of sugarcane cytogenetics. In this study, FCM probes were conducted to estimate 2C DNA content in 175 Saccharum genotypes embracing phenotypically contrasting canes (commercial cultivars, energy‐cane hybrids, and a Saccharum spontaneum accession) and full‐sib sugarcane families. FCM‐mediated DNA content estimates exhibited high accuracy in predicting chromosome numbers, offering a time‐efficient alternative to classical cytogenetic procedure. Low‐magnitude correlations detected between DNA content and phenotypic traits suggest that indirect selection of agronomic traits using FCM‐mediated DNA content estimates may not be efficient. DNA content variation patterns in sugarcane families varied according to the crossing, and revealed transgressive variations associated with rampant aneuploidy. Altogether, the findings uncover that loss or gain of genetic material constitutes a source of high genetic variability that may substantially impact on crop breeding.

Genomic linkages dictate cancer evolution

Aneuploidy, a state of chromosome imbalance frequently found in cancer, results in convoluted cancer genomes. Here, Kuzmin and colleagues1 identify how the aneuploid genome in triple-negative breast cancer is being shaped by unique genome network interactions.

Establishment of an Efficient Sugarcane Transformation System via Herbicide-Resistant CP4-EPSPS Gene Selection

Sugarcane (Saccharum spp.), a major cash crop that is an important source of sugar and bioethanol, is strongly influenced by the impacts of biotic and abiotic stresses. The intricate polyploid and aneuploid genome of sugarcane has shown various limits for conventional breeding strategies. Nonetheless, biotechnological engineering currently offers the best chance of introducing commercially significant agronomic features. In this study, an efficient Agrobacterium-mediated transformation system that uses the herbicide-resistant CP4-EPSPS gene as a selection marker was developed. Notably, all of the plants that were identified by PCR as transformants showed significant herbicide resistance. Additionally, this transformation protocol also highlighted: (i) the high yield of transgenic lines from calli (each gram of calli generated six transgenic lines); (ii) improved selection; and (iii) a higher transformation efficiency. This protocol provides a reliable tool for a routine procedure for the generation of resilient sugarcane plants.

Super-Resolution Microscopy Unveils Synergistic Structural Changes of Organelles Upon Point Mutation.

Ethyl methanesulphonate (EMS), is a widely used chemical mutagen that causes high-frequency germline null mutation by inserting an alkyl group into the nucleotide guanine in eukaryotic cells. The effect of EMS on the dynamics of the aneuploid genome, increased cellular instability, and carcinogenicity in relation to benign and malignant tumors are reported, but the molecular level understanding of morphological changes of higher-order chromatin structure has poorly been understood. This is due to a lack of sufficient resolution in conventional microscopic techniques to see small structures below the diffraction limit. Here, using super-resolution radial fluctuation, a largely fragmented, decompaction, and less dense heterochromatin structure upon EMS treatment to HEK 293A cells without any change in nuclear DNA domains is observed. This result suggests an early stage of carcinogenicity happened due to the point mutation. In addition, the distinct structural changes with an elongated morphology of lysosomes are also observed. On the other hand, fragmented and increased heterogeneous populations with an increased cytoplasmic occupancy of mitochondria are observed.

Anaplastic transformation in thyroid cancer revealed by single cell transcriptomics.

The deadliest anaplastic thyroid cancer (ATC) often transforms from indolent differentiated thyroid cancer (DTC); however, the complex intra-tumor transformation process is poorly understood. We investigated an anaplastic transformation model by dissecting both cell lineage and cell fate transitions using single cell transcriptomes and genetic alteration data from patients with different subtypes of thyroid cancer. The resulting spectrum of ATC transformation included stress-responsive DTC cells, inflammatory ATC cells (iATCs), mitotic-defective ATC cells and extended all the way to mesenchymal ATC cells (mATCs). Further, our analysis identified two important milestones: 1) a diploid stage, where iATC cells were diploids with inflammatory phenotypes, and 2) an aneuploid stage, where mATCs gained aneuploid genomes and mesenchymal phenotypes producing excessive collagens and collagen-interacting receptors. In parallel, cancer-associated-fibroblasts showed strong interactions among mesenchymal cell-types, macrophages shifted from M1 to M2 states, and T cells reprogrammed from cytotoxic to exhausted states, highlighting new therapeutic opportunities for ATC.

Abstract 314: Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer

Abstract The high incidence of colon cancer (CC) worldwide is a major health concern. Although surgery and adjuvant chemotherapy are effective to a high extent in lymph-node negative colon cancers, there are still some 10-15% of patients that show disease relapse within the next 5-year period after intended curative surgery. We hypothesize that genomic instability and intratumor heterogeneity fuel the formation of subclonal populations in the primary tumor that might promote metastasis. In the current study, we assessed the presence of intratumor heterogeneity in CC using multiplex interphase FISH (miFISH) and whole-exome sequencing (WES) to evaluate samples obtained from primary stage II colon tumors and their patient-matched associated liver metastases using multi-region sampling (N=9). In combination with genome-wide copy-number changes obtained from WES analysis, miFISH allowed the simultaneous quantification of copy-numbers for nine CC relevant genes and a centromeric ploidy control probe in intact tumor nuclei derived from archival patient material. Our preliminary data on the four cases analyzed so far, revealed high similarities regarding copy-number changes and ploidy between primary tumor and metastatic samples for three of the cases. While two of these cases showed either triploid or highly aneuploid genomes with different degrees of genomic instability and subclonality among them, the third case displayed a diploid content for both matched lesions. Intriguingly, as seen by miFISH and WES, loss of CDX2 in the primary tumor might have originated the main clonal population giving rise to metastasis in this latter case. Conversely, the fourth case analyzed exhibited a triploid baseline for the primary tumor while the metastasis revealed a diploid population. Interestingly, despite higher absolute copy numbers in the primary tumor, there was an overlap of five of the gains and losses observed for the major clone populations of the tumor and metastasis by miFISH. However, two of the highly clonal gains observed in the tumor, namely the EGFR and MYC gain, were not present in the metastasis. Altogether, in addition to the ploidy change, results indicated major discrete copy-number changes from primary tumor to metastasis in this patient. In summary, we show here that the combined analysis of WES and miFISH is able to describe the subclonal composition of copy-number alterations in primary tumors and their patientmatched liver metastases. Future perspectives also include the assessment of subclonality affecting single-nucleotide variants to complete the genomic landscape of tumor evolution and clonal development in this patient cohort with the goal to better understand the nature of these early metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Sara Lahoz, Veronica Pablo-Fontecha, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 314.

Abstract 2205: KRTAP 2-3 is a novel potential biomarker of cells in the polyaneuploid cancer cell (PACC) state to predict cancer recurrence

Abstract Prostate cancer (PCa) is the second leading cause of death in American men. PCa death is primarily attributed to therapeutic resistance. Our group and others have recently discovered a stress-induced adaptive cell state that plays a key role in therapy resistance: the polyaneuploid cancer cell (PACC) state. Cells in the polyaneuploid cancer cell (PACC) state are characterized by large cell size and polyploidization of the baseline aneuploid genome. Cells in the PACC state (e.g., pleomorphic giant cells, polyploid giant cancer cells, osteoclast like cells) have been reported in patient tumor specimens of multiple cancer types. We recently reported that in primary tumor specimens of PCa patients undergoing radical prostatectomy with curative intent, the number of cells in the PACC state is a statistically significant negative prognostic factor for metastasis-free survival (data adjusted for CAPRA-S). These data highlight the promise of the PACC state as a predictive biomarker for recurrence. In our previous work, cells in the PACC state were identified and quantified through EpCAM immunohistochemistry (IHC) to visualize cell membranes, manually including cells with &gt3x increased nuclear area compared to adjacent non-polyploid cancer cells. However, this method is labor intensive and highly subjective. There are currently no biomarkers to distinguish cells in the PACC state. To identify candidate PACC state biomarkers, we performed RNA sequencing to identify differentially expressed genes. Across two independent RNA sequencing datasets of PC3 cells induced to enter the PACC state with IC50 cisplatin or IC90 docetaxel, the most differentially expressed gene was KRTAP2-3 (Log2FC=4.3 and 5.51, respectively). The Keratin Associated Protein 2-3 (KRTAP2-3) is encoded by one of the homologous KRTAP genes classified by high sulfur content and is primarily expressed in the cortex of the hair follicle. It has not been previously implicated in PCa or in chemotherapy resistance. Due to its differential RNA expression in the PACC state and its low expression in most normal tissues, we hypothesize that KRTAP2-3 can be used as a biomarker to distinguish cells in the PACC state in patient tumor samples. To test this directly, we optimized RNA in situ-hybridization (RNAish) for KRTAP2-3. In initial experiments utilizing PC3 cell line plugs, we found that the KRTAP2-3 RNAish signal successfully distinguished cells in the PACC state (50.6 average punctae/cell) from non-polyploid cells (0.19 average punctae/cell) reflecting 96% percent accuracy using sensitivity and specificity metrics. We have further confirmed positive KRTAP2-3 signal on PC3 cells induced to access the PACC state in response to cisplatin, docetaxel, and etoposide treatment. Future work will evaluate the utility of the KRTAP2-3 gene as a prognostic biomarker that marks cells in the PACC state within PCa patient tissue microarrays. Citation Format: Sabrina A. Mendez, Reema Sharma, Kenneth J. Pienta, Sarah R. Amend. KRTAP 2-3 is a novel potential biomarker of cells in the polyaneuploid cancer cell (PACC) state to predict cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2205.

Abstract 5988: A non-canonical CDK9 complex mediates endocycling in polyaneuploid cancer cell (PACC) state

Abstract Once prostate cancer has spread from its primary site, treatment is limited to systemically administered therapy. While tumors initially respond to these therapies, eventually, all therapies fail, and the tumor recurs. Ours and others' recent studies have shown the presence of a universal mechanism of resistance: accession of the polyaneuploid cancer cell (PACC) state. The PolyAneuploid Transition (PAT) is initiated when a cancer cell is subjected to external stress, including therapy (we have tested three classes of chemotherapy and radiation), and accesses an alternative non-mitotic cell cycle known as endocycling, resulting in a greater-than-G2 polyploidization of the aneuploid genome (polyaneuploidy). While in this pro-survival PACC state, we have shown that cells are resistant to all forms of cytotoxic therapy. Initial RNA sequencing of cells that have accessed the PACC state in response to multiple classes of chemotherapy, including docetaxel and cisplatin, demonstrate a remodeled transcriptional profile that is distinct from control cultures. We hypothesize that accession of the resistance-mediating PACC state requires transcriptional remodeling. Our data show that cells in the PACC state had increased overall transcriptional activity. The multi protein complex pTEFB (positive transcription elongation factor) is a critical driver of RNA Polymerase II function and is composed of multiple subunits, including CDK9, a cyclin regulatory subunit, and a transcription factor. CDK9 phosphorylation was significantly elevated during accession and persistence in the PACC state. Genetic inhibitionof CDK9 by siRNA led to decreased expression of cyclin A which plays a role in S phase, both in the mitoticcycle and in the PACC endocycle. Interestingly, despite increased CDK9 levels, known CDK9 pTEFb complex members cyclin T1 and BRD4 were both decreased in cells in the PACC state. We treated cancer cells induced to enter the PACC state upon exposure to cisplatin with pharmaceutical inhibitors of CDK9, Flavopiridol and Fadraciclib. Combination of CDK9 inhibition with chemotherapy showed a suppressive effect on the PACC population. Our findings therefore suggest that a non-canonical CDK9 complex could be critical role for the endocycling to access and maintain the PACC state and represents a therapeutic candidate to eliminate the resistance-mediating PACC state. Citation Format: Cheng-fan Lee, Michael Loycano, Luke LOFTUS, Laurie Kostecka, Melvin Li, Sarah Amend, Kenneth Pienta. A non-canonical CDK9 complex mediates endocycling in polyaneuploid cancer cell (PACC) state. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5988.

THE BLASTULATION RATE AND THE RATIO OF 1PN/2PN EMBRYOS ARE ASSOCIATED WITH THE COHORT OF EMBRYOS PLOIDY RATIO

Blastulation is defined as the formation of the blastocyst from a pre compaction embryo and is an early stage in embryonic development. While an aneuploid genome is not incompatible with blastocyst formation, several trials have shown that aneuploidy does affect blastulation. We sought to study the association between the blastulation rate, the presence of 1 pro nuclei (1PN) embryos and the number of blastocysts available for biopsy on their ploidy.

Abstract 1795: Molecular vulnerabilities of the polyaneuploid cancer cell state

Abstract Once cancer has metastasized it remains incurable owing to resistance to nearly all systemic therapies. Classical views of therapeutic resistance include tumor heterogeneity and genetic instability, but our group and others have identified an unappreciated cancer cell phenotype, the polyaneuploid cancer cell (PACC) state, that provides an alternative model. The PACC state is induced upon exposure to a variety of applied stressors, including multiple different classes of chemotherapy, and is characterized by an enlarged cell size, polyploidy of the typically aneuploid cancer genome, and the ability to repopulate tissue through depolyploidization. We have demonstrated that PACCs are present in a variety of cell lines, arise in response to a multitude of stressors in vitro, and are resistant to cisplatin, docetaxel and Ipatasertib treatment. Thus, we hypothesize that the PACC state is driving cancer’s therapeutic resistance to nearly all available treatment regimens and enabling cancer relapse. Elimination of the PACC state is essential to prevent cancer recurrence, and ultimately enable a cure for cancer.To identify candidate drug targets specific to the PACC state, we performed RNA sequencing, NanoString mRNA panels, bulk proteomics and cell surface proteomics of PACCs versus their parental cell line. PACCs were induced from prostate cancer lines PC3 and LNCaP, and breast cancer line MDAMB231 with both cisplatin and docetaxel treatment. Initial datasets with PC3 cells found that 1013 genes are enriched (greater than 1.5 Log2 Fold Change) in cisplatin induced PACCs while 88 genes are enriched in docetaxel induced PACCs. 22 genes were enriched with both chemotherapy treatments implying that there are genes common to the PACC state. NanoString mRNA and proteomic analysis further validated a subset of the overlapping genes, including ATF3, BIRC3, IL6, and NOX5.Our existing data reveals that there are molecular vulnerabilities of the PACC state that can be manipulated for therapeutic elimination. We will further validate candidate genes across numerous stressors and cell lines and then assess the PACC state’s dependence on these pathways through inhibition and engineered cell lines. Information from these studies will directly lead to development of a therapeutic strategy specifically targeting the PACC state, with the intention of eliminating cancer resistance. Citation Format: Luke Loftus. Molecular vulnerabilities of the polyaneuploid cancer cell state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1795.

Abstract 123: Reactive oxygen species (ROS) as mediators of the cancer cell’s transition into the polyaneuploid cancer cell (PACC) state

Abstract Metastatic cancer is lethal and incurable due to the emergence of therapeutic resistance, resulting in the deaths of 10 million people each year. Our data suggests that therapeutic resistance is mediated by stress-induced transition into a protective polyaneuploid cancer cell (PACC) state. The PACC state is characterized by large size, polyploidization of the original aneuploid genome, and pause in cell cycle. Once released from stress, some PACCs survive to repopulate a non-polyploid, resistant cancer cell population. Using Nanostring technology to profile cisplatin-induced PACCs in prostate cancer PC3 and breast cancer MDA-MB-231 cells, we observed significant increase in gene expression related to reactive oxygen species (ROS) and the DNA damage response (DDR). We hypothesize that ROS mediate a cancer cell’s ability to enter the PACC state to survive chemotherapy.ROS are produced as byproducts of cellular respiration and are present in all cells. ROS radicals are damaging to cellular macromolecules, particularly DNA. Cancer cells have evolved to withstand high ROS, but the increase in radicals contribute to genomic instability. ROS-induced DNA damage activates the DDR leading to four possible fates: continued cycling, DNA repair, apoptosis, or cell cycle arrest. Therapy significantly increases DNA damage while also increasing total ROS levels. At this level of DNA damage, the likely cell fates are death or cycle arrest. We induced the PACC state in PC3 cells with cisplatin and monitored ROS during PACC formation (24, 48, and 72 hours) using fluorescent microscopy and flow cytometry techniques. After 72 hours, the only surviving cells are in the PACC state. We found a step wise increase in the amount of ROS per cell across treatment time. By 72 hours, total ROS is 7-fold higher in PACCs compared to the parental control. PACCs are substantially larger than parental controls, so cell size must be considered. After adjusting for unit area, we found treated cells have a higher level of ROS compared to parental cells. The greatest increase in ROS per area was seen at 24hrs, suggesting there is an initial spike in ROS at the introduction of therapy. This suggests that the ability to enter the PACC state may be due to cell cycle arrest as a response to the extensive ROS caused by therapy. To assess cells that survive therapy in the PACC stateand produce resistant progeny, we induced PACC formation, then released therapy and monitored ROS for 10 days. Adjusted for area, ROS levels remained stable and elevated compared to parental control for 10 days following therapy release. The increase in ROS per unit area shows high ROS is not simply due to increased size. The stability over time suggests PACCs are able to manage ROS levels to prevent death by oxidative stress. Future work will evaluate PACCs induced by non-platinum chemotherapies, as well as, in additional cell lines. Citation Format: Morgan D. Kuczler, Kenneth J. Pienta, Sarah R. Amend. Reactive oxygen species (ROS) as mediators of the cancer cell’s transition into the polyaneuploid cancer cell (PACC) state [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 123.

Aneuploidy influences the gene expression profiles in Saccharomyces pastorianus group I and II strains during fermentation.

The lager yeasts, Saccharomyces pastorianus, are hybrids of Saccharomyces cerevisiae and Saccharomyces eubayanus and are divided into two broad groups, Group I and II. The two groups evolved from at least one common hybridisation event but have subsequently diverged with Group I strains losing many S. cerevisiae chromosomes while the Group II strains retain both sub-genomes. The complex genomes, containing orthologous alleles from the parental chromosomes, pose interesting questions regarding gene regulation and its impact on the fermentation properties of the strains. Superimposed on the presence of orthologous alleles are complexities of gene dosage due to the aneuploid nature of the genomes. We examined the contribution of the S. cerevisiae and S. eubayanus alleles to the gene expression patterns of representative Group I and II strains during fermentation. We show that the relative expression of S. cerevisiae and S. eubayanus orthologues is positively correlated with gene copy number. Despite the reduced S. cerevisiae content in the Group I strain, S. cerevisiae orthologues contribute to biochemical pathways upregulated during fermentation which may explain the retention of specific chromosomes in the strain. Conversely, S. eubayanus genes are significantly overrepresented in the upregulated gene pool in the Group II strain. Comparison of the transcription profiles of the strains during fermentation identified both common and unique gene expression patterns, with gene copy number being a dominant contributory factor. Thus, the aneuploid genomes create complex patterns of gene expression during fermentation with gene dosage playing a crucial role both within and between strains.

The Dynamic Instability of the Aneuploid Genome.

Proper partitioning of replicated sister chromatids at each mitosis is crucial for maintaining cell homeostasis. Errors in this process lead to aneuploidy, a condition in which daughter cells harbor genome imbalances. Importantly, aneuploid cells often experience DNA damage, which in turn could drive genome instability. This might be the product of DNA damage accumulation in micronuclei and/or a consequence of aneuploidy-induced replication stress in S-phase. Although high levels of genome instability are associated with cell cycle arrest, they can also confer a proliferative advantage in some circumstances and fuel tumor growth. Here, we review the main consequences of chromosome segregation errors on genome stability, with a special focus on the bidirectional relationship between aneuploidy and DNA damage. Also, we discuss recent findings showing how increased genome instability can provide a proliferation improvement under specific conditions, including chemotherapeutic treatments.

Interaction of Male Specific Lethal complex and genomic imbalance on global gene expression in Drosophila

The inverse dosage effect caused by chromosome number variations shows global consequences in genomic imbalance including sexual dimorphism and an X chromosome-specific response. To investigate the relationship of the MSL complex to genomic imbalance, we over-expressed MSL2 in autosomal and sex chromosomal aneuploids, and analyzed the different transcriptomes. Some candidate genes involved in regulatory mechanisms have also been tested during embryogenesis using TSA-FISH. Here we show that the de novo MSL complex assembled on the X chromosomes in females further reduced the global expression level on the basis of 2/3 down-regulation caused by the inverse dosage effect in trisomy through epigenetic modulations rather than induced dosage compensation. Plus, the sexual dimorphism effect in unbalanced genomes was further examined due to the pre-existing of the MSL complex in males. All these results demonstrate the dynamic functions of the MSL complex on global gene expression in different aneuploid genomes.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

Somatic Sex: On the Origin of Neoplasms With Chromosome Counts in Uneven Ploidy Ranges

Stable aneuploid genomes with nonrandom numerical changes in uneven ploidy ranges define distinct subsets of hematologic malignancies and solid tumors. The idea put forward herein suggests that they emerge from interactions between diploid mitotic and G0/G1 cells, which can in a single step produce all combinations of mono-, di-, tri-, tetra- and pentasomic paternal/maternal homologue configurations that define such genomes. A nanotube-mediated influx of interphase cell cytoplasm into mitotic cells would thus be responsible for the critical nondisjunction and segregation errors by physically impeding the proper formation of the cell division machinery, whereas only a complete cell fusion can simultaneously generate pentasomies, uniparental trisomies as well as biclonal hypo- and hyperdiploid cell populations. The term “somatic sex” was devised to accentuate the similarities between germ cell and somatic cell fusions. A somatic cell fusion, in particular, recapitulates many processes that are also instrumental in the formation of an abnormal zygote that involves a diploid oocyte and a haploid sperm, which then may further develop into a digynic triploid embryo. Despite their somehow deceptive differences and consequences, the resemblance of these two routes may go far beyond of what has hitherto been appreciated. Based on the arguments put forward herein, I propose that embryonic malignancies of mesenchymal origin with these particular types of aneuploidies can thus be viewed as the kind of flawed somatic equivalent of a digynic triploid embryo.

Leishmania Sexual Reproductive Strategies as Resolved through Computational Methods Designed for Aneuploid Genomes.

A cryptic sexual reproductive cycle in Leishmania has been inferred through population genetic studies revealing the presence of hybrid genotypes in natural isolates, with attempts made to decipher sexual strategies by studying complex chromosomal inheritance patterns. A more informative approach is to study the products of controlled, laboratory-based experiments where known strains or species are crossed in the sand fly vector to generate hybrid progeny. These hybrids can be subsequently studied through high resolution sequencing technologies and software suites such as PAINT that disclose inheritance patterns including ploidies, parental chromosome contributions and recombinations, all of which can inform the sexual strategy. In this work, we discuss the computational methods in PAINT that can be used to interpret the sexual strategies adopted specifically by aneuploid organisms and summarize how PAINT has been applied to the analysis of experimental hybrids to reveal meiosis-like sexual recombination in Leishmania.

Abstract PR06: Stabilising selection causes grossly altered but stable karyotypes in metastatic colorectal cancer

Abstract Aneuploidy, defined as the loss and gain of whole and part chromosomes, is a near-ubiquitous feature of cancer genomes, and has clinical relevance as a prognostic marker and potential therapeutic target. We have previously shown that in colorectal cancer (CRC), aneusomies largely occur upon the transition from benign adenoma to invasive carcinoma (Cross et al., 2018), however the temporal evolutionary dynamics that select for aneuploidy throughout CRC progression remain broadly uncharacterised. We performed multi-region genomic analysis of 755 samples from a total of 167 patients with colorectal-derived neoplastic lesions that cross-sectionally represent the distinct stages of CRC progression, and longitudinally tracked 22 individual CRC tumours through metastasis and treatment. Genetic diversity measures revealed that although adenomas exhibited low levels of aneuploidy (8.5% genome altered), they harboured high intra-tumour heterogeneity (ITH), whereas cancers had significantly higher aneuploidy (27.2% genome altered) but proportionally lower ITH (p=0.0017), indicating that progression to malignancy is through a genetic bottleneck that suppresses diversity. We generated genome-wide copy number profiles from individual CRC glands, and showed that glands from the same tumour had similar karyotypes, yet displayed ongoing instability at the single cell level. Remarkably, we found that aneuploid genomes were broadly stable in metastatic lesions, even after chemo- or targeted therapies, and in recurrences detected many years after the primary diagnosis. Computational modelling of the dynamics of aneusomy accrual during CRC progression to metastasis was consistent with stabilising selection acting to ‘trap’ cancer cell genomes on a fitness peak defined by the optimal patterns of aneuploidy. These data show that although a genetic bottleneck underlies the initial progression from adenoma to CRC, further evolution of CRC is constrained largely by stabilising selection, even through metastatic dissemination and therapeutic resistance. References Cross, W., Kovac, M., Mustonen, V., Temko, D., Davis, H., Baker, A.-M., … Tomlinson, I. P. M. (2018). The evolutionary landscape of colorectal tumorigenesis. Nature Ecology & Evolution, 2(10), 1661–1672. https://doi.org/10.1038/s41559-018-0642-z Citation Format: Salpie Nowinski, William Cross, Maximilian Mossner, George Cresswell, Abhirup Banerjee, Marc Williams, Laura Gay, Ann-Marie Baker, Binxgin Lu, Christopher Kimberley, Hayley Davis, Nik Matthews, Miangela M. Lacle, Ian P.M. Tomlinson, Marnix Jansen, Alan Watson, Chris Barnes, Nicholas A. Wright, John Bridgewater, Manuel Rodriguez-Justo, Hemant Kocher, Simon J. Leedham, Andrea Sottoriva, Trevor A. Graham. Stabilising selection causes grossly altered but stable karyotypes in metastatic colorectal cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR06.