The extent of whole-genome copy number alterations predicts aggressive features in primary melanomas

307 Background: AR targeted therapies in combination with PARP inhibitors have recently shown efficacy in mCRPC patients with specific DNA repair gene mutations in metastatic tissue biopsies. Homologous recombination DNA repair deficiencies (HRD), associated with response to PARP inhibitors, can also be assessed by genomic instability/scarring. Accurate genomic scarring measurement in tumors can be confounded by intra-tumor heterogeneity and/or non-tumor genome contamination. To better identify PARPi sensitivity in mCRPC patients, we developed a genomic instability and scarring assay starting from single CTCs. Methods: VCaP, LnCaP or PC3 cell lines were spiked into healthy donor blood. Individual spiked cells were identified and recovered for genomic analysis using the standard Epic CTC assay. Post recovery, cells were lysed, whole genome amplified, constructed into shotgun libraries and sequenced to ~2M 2x150bp PE reads. Following alignment, whole genome copy number and instability analysis was performed to identify large scale transitions (LST, n of chromosomal breaks between adjacent regions of at least 10 Mb), % of genome altered (percentage of 1Mbp bins with copy number alterations), as well as specific tumor suppressor/oncogene copy number alterations. The association of PTEN loss with increases in genomic instability and scaring was performed. Results: Loss of PTEN function was previously shown to be associated with genomic instability. Our assessment of PTEN deletion was confirmed in PC3, while at least one genomic copy was observed in LnCaP and VCaP. The number of LSTs and % of genome altered was higher in PC3 (n = 19 +/- 3; 9.3% +/- 2.6%) than both VCaP and LnCap (n = 8 +/- 2; 6.1% +/- 0.4%). Conclusions: The association of higher genomic instability in a PTEN null cell line (PC3) vs. those with either heterozygous (LnCaP) or wild type (VCaP) PTEN status, matches published reports associating PTEN loss with increased genomic instability. Although, PC3 only demonstrates mild PARPi sensitivity in vitro, the detection of increased genomic scarring vs. cells with at least 1 functional PTEN allele confirms the assays ability to quantify genome instability at the single cell level from CTCs in a liquid biopsy.

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Nuclear-cytoplasmic balance: whole genome duplications induce elevated organellar genome copy number

10.1101/2021.06.08.447629 ◽

2021 ◽

Author(s):

Matheus Fernandes Gyorfy ◽

Emma R Miller ◽

Justin L Conover ◽

Corrinne E Grover ◽

Jonathan F Wendel ◽

...

Keyword(s):

Copy Number ◽

Nuclear Genome ◽

Plant Genome ◽

Whole Genome ◽

Genome Copy ◽

Relative Copy Number ◽

Organellar Genome ◽

Plastid Genomes ◽

Copy Numbers ◽

Genome Copy Number

The plant genome is partitioned across three distinct subcellular compartments: the nucleus, mitochondria, and plastids. Successful coordination of gene expression among these organellar genomes and the nuclear genome is critical for plant function and fitness. Whole genome duplication events (WGDs) in the nucleus have played a major role in the diversification of land plants and are expected to perturb the relative copy number (stoichiometry) of nuclear, mitochondrial, and plastid genomes. Thus, elucidating the mechanisms whereby plant cells respond to the cytonuclear stoichiometric imbalance that follow WGDs represents an important yet underexplored question in understanding the evolutionary consequences of genome doubling. We used droplet digital PCR (ddPCR) to investigate the relationship between nuclear and organellar genome copy numbers in allopolyploids and their diploid progenitors in both wheat and Arabidopsis. Polyploids exhibit elevated organellar genome copy numbers per cell, largely preserving the cytonuclear stoichiometry observed in diploids despite the change in nuclear genome copy number. To investigate the timescale over which cytonuclear stoichiometry may respond to WGD, we also estimated organellar genome copy number in Arabidopsis synthetic autopolyploids and in a haploid-induced diploid line. We observed corresponding changes in organellar genome copy number in these laboratory-generated lines, indicating that at least some of the cellular response to cytonuclear stoichiometric imbalance is immediate following WGD. We conclude that increases in organellar genome copy numbers represent a common response to polyploidization, suggesting that maintenance of cytonuclear stoichiometry is an important component in establishing polyploid lineages.

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