High resolution copy number inference in cancer using short-molecule nanopore sequencing.

Genome copy number is an important source of genetic variation in health and disease. In cancer, Copy Number Alterations (CNAs) can be inferred from short-read sequencing data, enabling genomics-based precision oncology. Emerging Nanopore sequencing technologies offer the potential for broader clinical utility, for example in smaller hospitals, due to lower instrument cost, higher portability, and ease of use. Nonetheless, Nanopore sequencing devices are limited in the number of retrievable sequencing reads/molecules compared to short-read sequencing platforms, limiting CNA inference accuracy. To address this limitation, we targeted the sequencing of short-length DNA molecules loaded at optimized concentration in an effort to increase sequence read/molecule yield from a single nanopore run. We show that short-molecule nanopore sequencing reproducibly returns high read counts and allows high quality CNA inference. We demonstrate the clinical relevance of this approach by accurately inferring CNAs in acute myeloid leukemia samples. The data shows that, compared to traditional approaches such as chromosome analysis/cytogenetics, short molecule nanopore sequencing returns more sensitive, accurate copy number information in a cost effective and expeditious manner, including for multiplex samples. Our results provide a framework for short-molecule nanopore sequencing with applications in research and medicine, which includes but is not limited to, CNAs

Replication timing maintains the global epigenetic state in human cells

ACKNOWLEDGMENTS We thank R. Didier and B. Alexander of the FSU Flow Cytometry and Confocal Microscopy Facilities for their help with flow cytometry and fluorescence-activated cell sorting for this project. Thanks to A. Brown of the FSU Biological Science Core Labs and to Y. Yang and C. Vied of the FSU Translational Labs. Thanks to S. R. Westermann of SCIGRAPHIX for generating the model figure. Thanks to B. van Steensel, J. Phillips-Cremins, and P. Fraser for critical reading of the manuscript. Funding: This work was supported by NIH grant GM083337 to D.M.G., GM035463 to V.G.C., and GM085354 to D.M.G., S.D., and V.G.C. D.L. is supported by the Hong Kong Research Grant Council (ECS 26104216). T.B. is supported by the William C. and Joyce C. O’Neil Charitable Trust, Memorial Sloan Kettering Single Cell Sequencing InitiativePeer reviewedPostprin

Senescence induction dictates response to chemo- and immunotherapy in preclinical models of ovarian cancer

High-grade serous ovarian carcinoma (HGSOC) is a cancer with dismal prognosis due to the limited effectiveness of existing chemo- and immunotherapies. To elucidate mechanisms mediating sensitivity or resistance to these therapies, we developed a fast and flexible autochthonous mouse model based on somatic introduction of HGSOC-associated genetic alterations into the ovary of immunocompetent mice using tissue electroporation. Tumors arising in these mice recapitulate the metastatic patterns and histological, molecular, and treatment response features of the human disease. By leveraging these models, we show that the ability to undergo senescence underlies the clinically observed increase in sensitivity of homologous recombination (HR)-deficient HGSOC tumors to platinum-based chemotherapy. Further, cGas/STING-mediated activation of a restricted senescence-associated secretory phenotype (SASP) was sufficient to induce immune infiltration and sensitize HR-deficient tumors to immune checkpoint blockade. In sum, our study identifies senescence propensity as a predictor of therapy response and defines a limited SASP profile that appears sufficient to confer added vulnerability to concurrent immunotherapy and, more broadly, provides a blueprint for the implementation of electroporation-based mouse models to reveal mechanisms of oncogenesis and therapy response in HGSOC

Mcm2 hypomorph leads to acute leukemia or hematopoietic stem cell failure, dependent on genetic context

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2Cre ) have decreased Mcm2 expression and invariably develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL), due to 100-1000 kb deletions involving important tumor suppressor genes. To determine whether mice that were protected from pre-T LBL would develop non-T-cell malignancies, we used two approaches. Mice engrafted with Mcm2Cre/Cre Lin- Sca-1+ Kit+ hematopoietic stem/progenitor cells did not develop hematologic malignancy; however, these mice died of hematopoietic stem cell failure by 6 months of age. Placing the Mcm2Cre allele onto an athymic nu/nu background completely prevented pre-T LBL and extended survival of these mice three-fold (median 296.5 vs. 80.5 days). Ultimately, most Mcm2Cre/Cre ;nu/nu mice developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We identified recurrent deletions of 100-1000 kb that involved genes known or suspected to be involved in BCP-ALL, including Pax5, Nf1, Ikzf3, and Bcor. Moreover, whole-exome sequencing identified recurrent mutations of genes known to be involved in BCP-ALL progression, such as Jak1/Jak3, Ptpn11, and Kras. These findings demonstrate that an Mcm2Cre/Cre hypomorph can induce hematopoietic dysfunction via hematopoietic stem cell failure as well as a "deletor" phenotype affecting known or suspected tumor suppressor genes

Interactive analysis and quality assessment of single-cell copy-number variations

Single-cell sequencing is emerging as a critical technology for understanding the biology of cancer, neurons, and other complex systems. Here we introduce Ginkgo, a web platform for the interactive analysis and quality assessment of single-cell copy-number alterations. Ginkgo fully automates the process of binning, normalizing, and segmenting mapped reads to infer copy number profiles of individual cells, as well as constructing phylogenetic trees of how those cells are related. We validate Ginkgo by reproducing the results of five major single-cell studies, and discuss how it addresses the wide array of biases that affect single-cell analysis. We also examine the data characteristics of three commonly used single-cell amplification techniques: MDA, MALBAC, and DOP-PCR/WGA4 through comparative analysis of 9 different single-cell datasets. We conclude that DOP-PCR provides the most uniform amplification, while MDA introduces substantial biases into the analysis. Furthermore, given the same level of coverage, our results indicate that data prepared using DOP-PCR can reliably call CNVs at higher resolution than data prepared using either MALBAC or MDA. Ginkgo is freely available at http://qb.cshl.edu/ginkgo.Received November 11, 2014.Accepted November 12, 2014.© 2014, Published by Cold Spring Harbor Laboratory PressThis pre-print is available under a Creative Commons License (Attribution-NonCommercial-NoDerivs 4.0 International), CC BY-NC-ND 4.0, as described at http://creativecommons.org/licenses/by-nc-nd/4.0

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal alpha-toxin

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor kappa B signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor alpha-toxin. Naturally elicited antibodies against alpha-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to alpha-toxin in nonleukocytic cells.Peer reviewe

Ordered and deterministic cancer genome evolution after p53 loss

Although p53 inactivation promotes genomic instability1 and presents a route to malignancy for more than half of all human cancers2,3, the patterns through which heterogenous TP53 (encoding human p53) mutant genomes emerge and influence tumorigenesis remain poorly understood. Here, in a mouse model of pancreatic ductal adenocarcinoma that reports sporadic p53 loss of heterozygosity before cancer onset, we find that malignant properties enabled by p53 inactivation are acquired through a predictable pattern of genome evolution. Single-cell sequencing and in situ genotyping of cells from the point of p53 inactivation through progression to frank cancer reveal that this deterministic behaviour involves four sequential phases-Trp53 (encoding mouse p53) loss of heterozygosity, accumulation of deletions, genome doubling, and the emergence of gains and amplifications-each associated with specific histological stages across the premalignant and malignant spectrum. Despite rampant heterogeneity, the deletion events that follow p53 inactivation target functionally relevant pathways that can shape genomic evolution and remain fixed as homogenous events in diverse malignant populations. Thus, loss of p53-the 'guardian of the genome'-is not merely a gateway to genetic chaos but, rather, can enable deterministic patterns of genome evolution that may point to new strategies for the treatment of TP53-mutant tumours

Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance

Defining the heterogeneity landscape of pancreatic cancer copy number alterations.

e15776 Background: Pancreatic cancer (PDAC) is projected to become the second leading cause of cancer related mortality by 2030. Bulk whole genome sequencing studies of PDAC have characterized the landscape of clonal mutations and highlighted the prominence of copy number alterations (CNAs) in PDAC genomes. However, little is known with regards to the extent of sub-clonal heterogeneity of somatic CNAs and it is hypothesized that this heterogeneity is a contributing factor to the limited effectiveness of existing therapies. Methods: We retrieved absolute copy number information using bulk sparse whole genome sequencing of multi-region sampled PDAC samples as well as matching primary-metastasis tumor samples from over 100 patients. In addition, we analyzed copy number variations in a subset of these samples (n = 15) at single-cell resolution (~1000 cells in total). Results: We describe a detailed picture of sub-clonal CNAs genetic heterogeneity. Our results illustrate, among other findings, (1) extensive sub-clonal diversity of CNAs giving rise to many genetically unique sub-clonal cancer populations, (2) somatic mosaicism of chromosomal amplicons in single-cancer cells, (3) variation in the dosage of cancer genes, including the KRAS oncogene, in different tumor sub-clones and (4) somatic alterations, such as amplification of 8q11 containing the metastasis promoting gene IKBKB, associated with primary PDAC progression to liver metastasis. Conclusions: Our results offer an in-depth view of the sub-clonal heterogeneity of somatic CNAs in pancreatic cancer and illustrate ways in which such heterogeneity could lead to therapeutic resistance. </jats:p