Whole genome doubling (WGD) occurs early in tumorigenesis and generates genetically unstable tetraploid cells that fuel tumor development. Cells that undergo WGD (WGD+) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can subsequently be exploited therapeutically, is unclear. Using sequencing data from ~10,000 primary human cancer samples and essentiality data from ~600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent on spindle assembly checkpoint signaling, DNA replication factors, and proteasome function than WGD– cells. We also identify KIF18A, which encodes for a mitotic kinesin, as being specifically required for the viability of WGD+ cells. While loss of KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD– cells, its loss induces dramatic mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results reveal new strategies to specifically target WGD+ cancer cells while sparing the normal, non-transformed WGD– cells that comprise human tissue.

Paralog reduction, the loss of duplicate genes after whole genome duplication (WGD) is a pervasive process. Whether this loss proceeds gene by gene or through deletion of multi-gene DNA segments is controversial, as is the question of fractionation bias, namely whether one homeologous chromosome is more vulnerable to gene deletion than the other. As a null hypothesis, we first assume deletion events, on one homeolog only, excise a geometrically distributed number of genes with unknown mean mu, and these events combine to produce deleted runs of length l, distributed approximately as a negative binomial with unknown parameter r; itself a random variable with distribution pi(.). A biologically more realistic model requires deletion events on both homeologs distributed as a truncated geometric. We simulate the distribution of run lengths l in both models, as well as the underlying pi(r), as a function of mu, and show how sampling l allows us to estimate mu. We apply this to data on a total of 15 genomes descended from 6 distinct WGD events and show how to correct the bias towards shorter runs caused by genome rearrangements. Because of the difficulty in deriving pi(.) analytically, we develop a deterministic recurrence to calculate each pi(r) as a function of mu and the proportion of unreduced paralog pairs. This is based on a computing formula containing nested sums. The parameter mu can be estimated based on run lengths of single-copy regions. We then reduce the computing formulae, at least in the one-sided case, to closed form. This virtually eliminates computing time due to highly nested summations. We formulate a continuous version of the fractionation process, deleting line segments of exponentially distributed lengths in analogy to geometric distributed numbers of genes. We derive nested integrals and discover that the number of previously deleted regions to be skipped by a new deletion event is exactly geometrically distributed. We undertook a large simulation experiment to show how to discriminate between the gene-by-gene duplicate deletion model and the deletion of a geometrically distributed number of genes. This revealed the importance of the effects of genome size N, the mean of the geometric distribution, the progress towards completion of the fractionation process, and whether the data are based on runs of deleted genes or undeleted genes.

Paralog reduction, the loss of duplicate genes after whole genome duplication (WGD) is a pervasive process. Whether this loss proceeds gene by gene or through deletion of multi-gene DNA segments is controversial, as is the question of fractionation bias, namely whether one homeologous chromosome is more vulnerable to gene deletion than the other. As a null hypothesis, we first assume deletion events, on one homeolog only, excise a geometrically distributed number of genes with unknown mean mu, and these events combine to produce deleted runs of length l, distributed approximately as a negative binomial with unknown parameter r; itself a random variable with distribution pi(.). A biologically more realistic model requires deletion events on both homeologs distributed as a truncated geometric. We simulate the distribution of run lengths l in both models, as well as the underlying pi(r), as a function of mu, and show how sampling l allows us to estimate mu. We apply this to data on a total of 15 genomes descended from 6 distinct WGD events and show how to correct the bias towards shorter runs caused by genome rearrangements. Because of the difficulty in deriving pi(.) analytically, we develop a deterministic recurrence to calculate each pi(r) as a function of mu and the proportion of unreduced paralog pairs. This is based on a computing formula containing nested sums. The parameter mu can be estimated based on run lengths of single-copy regions. We then reduce the computing formulae, at least in the one-sided case, to closed form. This virtually eliminates computing time due to highly nested summations. We formulate a continuous version of the fractionation process, deleting line segments of exponentially distributed lengths in analogy to geometric distributed numbers of genes. We derive nested integrals and discover that the number of previously deleted regions to be skipped by a new deletion event is exactly geometrically distributed. We undertook a large simulation experiment to show how to discriminate between the gene-by-gene duplicate deletion model and the deletion of a geometrically distributed number of genes. This revealed the importance of the effects of genome size N, the mean of the geometric distribution, the progress towards completion of the fractionation process, and whether the data are based on runs of deleted genes or undeleted genes.

Whole genome doubling (WGD) generates genetically unstable tetraploid cells that fuel tumorigenesis. Cells that undergo WGD must acquire adaptive characteristics to accommodate their tetraploid state, and these adaptations may confer unique vulnerabilities that can be exploited therapeutically. We analyzed the genomes of ~9,700 primary human cancer samples to uncover genetic alterations that are specifically enriched in WGD+ cancer cells. Through integrating our genetic analysis with gene essentiality data acquired from Project Achilles, we identified gene dependencies in WGD+ cells. Moreover, we identified genes that are essential for the viability of WGD+ cancer cells, but non-essential to non-transformed diploid cells. We demonstrated that the gene encoding for the mitotic kinesin KIF18A is dispensable for mitosis in diploid cells, but becomes critical for accurate chromosome segregation and viability in WGD+ cells, making it an attractive drug target. Collectively, this work revealed new strategies to specifically target WGD+ cancer cells, namely targeting the gene KIF18A, while sparing the normal diploid cells from which they arise. / 2022-06-04T00:00:00Z

Melanoma, a malignant neoplasm of melanocytes, is the most lethal form of skin cancer. A majority of melanomas are driven by activating mutations in the kinase BRAF, which drives cellular proliferation through constitutive stimulation of the mitogen-activated protein kinase (MAPK) signaling pathway. Intriguingly, expression of oncogenic BRAF alone in vivo is insufficient to promote melanoma; rather, its expression leads to the development of benign nevi (moles) comprised of growth-arrested melanocytes. The acquisition of additional genetic or epigenetic changes is therefore critical for melanocytes to evade arrest and drive melanomagenesis, however the identity of these changes remains incompletely understood. Here we demonstrate that expression of oncogenic BRAF leads to activation of the Hippo tumor suppressor pathway in vitro, which acts to limit melanocyte proliferation through the inhibition of the pro-growth transcriptional co-activators YAP and TAZ. Melanocyte-specific inactivation of Hippo signaling in vivo, via deletion of the Hippo kinases Lats1/2 alone, or in conjunction with oncogenic Braf expression, potently induces melanoma development in mice. Collectively, our data reveal that the Hippo tumor suppressor pathway represents an important barrier to melanoma development, and implicates YAP and TAZ as new therapeutic targets for the treatment of human melanoma.

Melanoma, the most lethal form of skin cancer, arises from altered cells in the melanocyte lineage, but the mechanisms by which these cells progress to melanoma are unknown. To understand the early cellular events that contribute to melanoma formation, we examined melanocytes in melanoma-prone zebrafish strains expressing BRAFV600E, the most common oncogenic form of the BRAF kinase that is mutated in nearly 50% of human melanomas. We found that, unlike wild-type melanocytes, melanocytes in transgenic BRAFV600Eanimals were binucleate and tetraploid. Furthermore, melanocytes in p53-deficient transgenic BRAFV600Eanimals exhibited 8N and greater DNA content, suggesting bypass of a p53-dependent arrest that stops cell cycle progression of tetraploid melanocytes. These data implicate tetraploids generated by increased BRAF pathway activity as contributors to melanoma initiation. Previous studies have used artificial means of generating tetraploids, raising the question of how these cells arise during actual tumor development. To gain insight into the mechanism by which BRAFV600E generates binucleate, tetraploid cells, we established an in vitro model by which such cells are generated following BRAFV600E expression. We demonstrate thatBRAFV600E-generated tetraploids arise via cytokinesis failure during mitosis due to reduced activity of the small GTPase RhoA. We also establish that oncogene-induced centrosome amplification in the G1/S phase of the cell cycle and subsequent increase in the activity of the small GTPase Rac1, partially contribute to this phenotype. These data are of significance as recent studies have shown that aneuploid progeny of tetraploid cells can be intermediates in tumor development, and deep sequencing data suggest that at least one third of melanomas and other solid tumors have undergone a whole genome doubling event during their progression. Taken together, our melanoma-prone zebrafish model and in vitro data suggest a role for BRAFV600E-inducedtetraploidy in the genesis of melanomas. To our knowledge, this is the first in vivo model showing spontaneous rise of tetraploid cells that can give rise to tumors. This novel role of the BRAF oncogene may contribute to tumorigenesis in a broader context.