Precision Medicine is an approach that takes into account the influence of individuals' genes, environment and lifestyle exposures to tailor interventions. Here, we describe the development of a robust precision cancer care platform, which integrates whole exome sequencing (WES) with a living biobank that enables high throughput drug screens on patient-derived tumor organoids. To date, 56 tumor-derived organoid cultures, and 19 patient-derived xenograft (PDX) models have been established from the 769 patients enrolled in an IRB approved clinical trial. Because genomics alone was insufficient to identify therapeutic options for the majority of patients with advanced disease, we used high throughput drug screening effective strategies. Analysis of tumor derived cells from four cases, two uterine malignancies and two colon cancers, identified effective drugs and drug combinations that were subsequently validated using 3D cultures and PDX models. This platform thereby promotes the discovery of novel therapeutic approaches that can be assessed in clinical trials and provides personalized therapeutic options for individual patients where standard clinical options have been exhausted.

Abstractp27 Kip1 is a candidate human tumour-suppressor protein, because it is able to inhibit cyclindependent kinases and block cell proliferation [1][2][3][4][5] . Abnormally low levels of the p27 protein are frequently found in human carcinomas, and these low levels correlate directly with both histological aggressiveness and patient mortality [6][7][8][9][10] . However, it has not been possible to establish a causal link between p27 and tumour suppression, because only rare instances of homozygous inactivating mutations of the p27 gene have been found in human tumours [11][12][13][14] . Thus, p27 Kip1 does not fulfill Knudson's 'two-mutation' criterion for a tumour suppressor gene 15 . Here we show that both p27 nullizygous and p27 heterozygous mice are predisposed to tumours in multiple tissues when challenged with g-irradiation or a chemical carcinogen. Therefore p27 is a multipletissue tumour suppressor in mice. Molecular analyses of tumours in p27 heterozygous mice show that the remaining wild-type allele is neither mutated nor silenced. Hence, p27 is haplo-insufficient for tumour suppression. The assumption that null mutations in tumour-suppressor genes are recessive excludes those genes that exhibit haplo-insufficiency.Tumour-suppressor proteins affect several cellular pathways, such as those controlling proliferation, apoptosis, differentiation and genomic integrity. Consequently, the identification of a tumour-suppressor gene is usually established genetically rather than by any particular functional criterion. The common operational definition of a tumoursuppressor gene requires the demonstration of mutations of both copies of a candidate gene in tumours 15 . Genetic and physical mapping of bi-allelic tumour specific mutations allows localization of tumour-suppressor genes, and provides evidence for a causal link between mutations in those genes and tumorigenesis. This approach has successfully demonstrated the tumour-suppressor function of the retinoblastoma protein (Rb), p53, INK4a, and others [16][17][18] . However, we show here that this definition is too restrictive because it excludes tumour suppressors such as p27 Kip1 that exhibit haplo-insufficiency.We studied the tumour-suppressor function of p27 Kip1 by examining the susceptibility of p27-deficient mice to tumorigenesis induced by two different DNA-damaging agents, gCorrespondence and requests for materials should be addressed to M.L.F. (mfero@fhcrc.org). -ethyl-N-nitrosourea (ENU). p27 −/− mice showed decreased tumour-free survival following g-irradiation compared with p27 +/+ controls (Fig. 1a). p27 +/− mice showed an intermediate sensitivity (see below). Thymic lymphomas were the most frequent tumours in wild-type mice (incidence 0.07), but their frequency was not significantly increased in p27 −/− animals (incidence 0.17). The increased mortality of p27-null mice was mainly due to the development of pituitary tumours and intestinal adenomas (Fig. 2). A separate group of mice was injected with a single dose of ENU, and again p27-nu...

Doxorubicin is one of the most important anti-cancer chemotherapeutic drugs, being widely used for the treatment of solid tumors and acute leukemias. The action of doxorubicin and other anthracycline drugs has been intensively investigated during the last several decades, but the mechanisms that have been proposed for cell killing remain disparate and controversial. In this review, we examine the proposed models for doxorubicin action from the perspective of the chromatin landscape, which is altered in many types of cancer due to recurrent mutations in chromatin modifiers. We highlight recent evidence for effects of anthracyclines on DNA torsion and chromatin dynamics that may underlie basic mechanisms of doxorubicin-mediated cell death and suggest new therapeutic strategies for cancer treatment.

Previous studies demonstrate that intrastriatal injections of fibrillar alpha-synuclein (α-syn) into mice induce Parkinson’s disease (PD)-like Lewy body (LB) pathology formed by aggregated α-syn in anatomically interconnected regions and significant nigrostriatal degeneration. The aim of the current study was to evaluate whether exogenous mouse α-syn pre-formed fibrils (PFF) injected into the striatum of rats would result in accumulation of LB-like intracellular inclusions and nigrostriatal degeneration. Sprague Dawley rats received unilateral intrastriatal injections of either non-fibrillized recombinant α-syn or PFF mouse α-syn in 1- or 2- sites and were euthanized at 30, 60 or 180 days post-injection (pi). Both non-fibrillized recombinant α-syn and PFF α-syn injections resulted in phosphorylated α-syn intraneuronal accumulations (i.e., diffuse Lewy neurite (LN)- and LB-like inclusions) with significantly greater accumulations following PFF injection. LB-like inclusions were observed in several areas that innervate the striatum, most prominently the frontal and insular cortices, the amygdala, and the substantia nigra pars compacta (SNpc). α-Syn accumulations co-localized with ubiquitin, p62, and were thioflavin-S-positive and proteinase-k resistant, suggesting PFF-induced pathology exhibits properties similar to human LBs. Although α-syn inclusions within the SNpc remained ipsilateral to striatal injection, we observed bilateral reductions in nigral dopamine neurons at the 180-day time point in both the 1- and 2-site PFF injection paradigms. PFF injected rats exhibited bilateral reductions in striatal dopaminergic innervation at 60 and 180 days and bilateral decreases in homovanillic acid; however, dopamine reduction was observed only in the striatum ipsilateral to PFF injection. Although the level of dopamine asymmetry in PFF injected rats at 180 days was insufficient to elicit motor deficits in amphetamine-induced rotations or forelimb use in the cylinder task, significant disruption of ultrasonic vocalizations was observed. Taken together, our findings demonstrate that α-syn PFF are sufficient to seed the pathological conversion and propagation of endogenous α-syn to induce a progressive, neurodegenerative model of α-synucleinopathy in rats.

SUMMARY Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding factors that induce driver mutations, and the processes that shape their selection during tumourigenesis. We performed whole-exome sequencing (WES) on adenomas from three mouse models of non-small cell lung cancer (NSCLC), induced by exposure to carcinogens (Methylnitrosourea (MNU) and Urethane), or by genetic activation of Kras (KrasLA2). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the KrasLA2 model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single nucleotide variants (SNVs), compared to only 6 in tumours from the KrasLA2 model. In contrast, the KrasLA2 tumours exhibited a significantly higher level of aneuploidy and copy number alterations (CNAs) compared to the carcinogen-induced tumours, suggesting that carcinogen and genetically-engineered models adopt different routes to tumour development. The wild type (WT) allele of Kras has been shown to act as a tumour suppressor in mouse models of NSCLC. We demonstrate that urethane-induced tumours from WT mice carry mostly (94%) Q61R Kras mutations, while those from Kras heterozygous animals carry mostly (92%) Q61L mutations, indicating a major role of germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C>T mutations at CpG sites. These data provide a basis for understanding the conclusions from human tumour genome sequencing that identified two broad categories based on relative frequency of SNVs and CNAs1, and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.

High-throughput technologies can now identify hundreds of candidate protein biomarkers for any disease with relative ease. However, because there are no assays for the majority of proteins and de novo immunoassay development is prohibitively expensive, few candidate biomarkers are tested in clinical studies. We tested whether the analytical performance of a biomarker identification pipeline based on targeted mass spectrometry would be sufficient for data-dependent prioritization of candidate biomarkers, de novo development of assays and multiplexed biomarker verification. We used a data-dependent triage process to prioritize a subset of putative plasma biomarkers from >1,000 candidates previously identified using a mouse model of breast cancer. Eighty-eight novel quantitative assays based on selected reaction monitoring mass spectrometry were developed, multiplexed and evaluated in 80 plasma samples. Thirty-six proteins were verified as being elevated in the plasma of tumor-bearing animals. The analytical performance of this pipeline suggests that it should support the use of an analogous approach with human samples.

No abstract

We have created two knock-in mouse models to study the mechanisms that regulate p27 in normal cells and cause misregulation of p27 in tumors: p27 S10A , in which Ser10 is mutated to Ala; and p27 CK − , in which point mutations abrogate the ability of p27 to bind cyclins and CDKs. These two mutant alleles identify steps in a pathway that controls the proteasomal degradation of p27 uniquely in quiescent cells: Dephosphorylation of p27 on Ser10 inhibits p27 nuclear export and promotes its assembly into cyclin-CDK complexes, which is, in turn, necessary for p27 turnover. We further show that Ras-dependent lung tumorigenesis is associated with increased phosphorylation on Ser10 and cytoplasmic mislocalization of p27. Indeed, we find that p27 S10A is refractory to Ras-induced cytoplasmic translocation and that p27 S10A mice are tumor resistant. Thus, phosphorylation of p27 on Ser10 is an important event in the regulation of the tumor suppressor function of p27.[Keywords: p27 Kip1 ; cyclin; CDK; tumor suppressor; tumorigenesis; Ras; lung cancer] Supplemental material is available at http://www.genesdev.org.