Functional identification and investigation of genes initiating chromosomal instability using CRISPR activation and high-throughput automated image analysis

Chromosomal instability (CIN), the dynamic state where cells experience increased structural and/or numerical chromosome segregation errors, is prevalent in cancer, where it contributes to aneuploidy and tumour evolution. Despite its profound consequences on human health, initiation of CIN in the early stages of tumourigenesis is not well understood. In this work multiple strategies were employed to investigate the effects of gene upregulation, as well as upregulation of the PIK3CA signalling pathway, a frequently altered pathway in many cancer types (Jamal-Hanjani et al. 2017; Teixeira et al. 2019). Combining CRISPR gene upregulation to model overexpression, high content imaging (HCS), and automated high-throughput image analysis, a pipeline was developed to screen for CIN and aneuploidy in a normal human cell line, RPE1. This provides a readout of micronuclei and centromere counts. Using this pipeline, upregulation of KIF11 was found to increase the proportion of cells exhibiting micronuclei, and cause significant deviation from the modal centromere count, indicating CIN and aneuploidy. Further investigation of this phenotype revealed that KIF11 upregulation causes spindle pole fragmentation, mitotic catastrophe, and chromosome congression defects. Centric and acentric lagging chromosomes were observed in cells that exhibited both normal and fragmented spindle poles. Mechanistically, KIF11 was shown to generate a force imbalance in the early stages, which could be partially rescued upon upregulation of HSET. MCF10A cell lines expressing PIK3CAH1047R at the endogenous genetic loci as a result of CRISPR genome editing were used to investigate the impact of increased signalling through the PIK3CA pathway on CIN, aneuploidy, and centrosome biology. This showed that PIK3CAH1047R increased the incidence of supernumerary centrosomes, and may play a role in structural CIN, but failed to identify any effect on numerical CIN. Finally, by chemically modulating PIK3CA activity, microtubule dynamics in response to PIK3CA pathway activation and inhibition were investigated

Reported waterborne outbreaks of gastrointestinal disease in Australia are predominantly associated with recreational exposure

Objective: To examine the frequency and circumstances of reported waterborne outbreaks of gastroenteritis in Australia. Method: Examination of data reported to OzFoodNet between 2001 and 2007. Results: During these seven years, 6,515 gastroenteritis outbreaks were reported to OzFoodNet, most of which were classifed as being transmitted person-to-person or from an unknown source. Fifty-four (0.83%) outbreaks were classifed as either 'waterborne' or 'suspected waterborne', of which 78% (42/54) were attributed to recreational water and 19% (10/54) to drinking water. Of the drinking water outbreaks, implicated pathogens were found on all but one occasion and included Salmonella sp. (fve outbreaks), Campylobacter jejuni (three outbreaks) and Giardia (one outbreak). Conclusions: There have been few waterborne outbreaks detected in Australia, and most of those reported have been associated with recreational exposure. However, there are difficulties in identifying and categorising gastroenteritis outbreaks, as well as in obtaining microbiological and epidemiological evidence, which can result in misclassifcation or underestimation of water-associated events. Implications: Gastroenteritis surveillance data show that, among reported waterassociated gastroenteritis outbreaks in Australia, recreational exposure is currently more common than a drinking water source. However, ongoing surveillance for waterborne outbreaks is important, especially as drought conditions may necessitate replacement of conventional drinking water supplies with alternative water sources, which could incur potential for new health risks

Modest increase of KIF11 exposes fragilities in the mitotic spindle causing chromosomal instability

Chromosomal instability (CIN), the process of increased chromosomal alterations, compromises genomic integrity and has profound consequences on human health. Yet, our understanding of the molecular and mechanistic basis of CIN initiation remains limited. We developed a high-throughput, single-cell image-based pipeline employing deep learning and spot counting models to detect CIN by automatically counting chromosomes and micronuclei. To identify CIN-initiating conditions, we used CRISPR activation in human diploid cells to upregulate, at physiologically-relevant levels, 14 genes that are functionally important in cancer. We found that upregulation of CCND1, FOXA1, and NEK2 resulted in pronounced changes in chromosome counts and KIF11 upregulation resulted in micronuclei formation. We identified KIF11-dependent fragilities within the mitotic spindle; increased KIF11 causes centrosome fragmentation, higher microtubule stability, lagging chromosomes or mitotic catastrophe. Our findings demonstrate that even modest average single gene expression changes in a karyotypically stable background are sufficient for initiating CIN by exposing fragilities of the mitotic spindle which can lead to a genomically-diverse cell population

Polyphenon E enhances the antitumor immune response in neuroblastoma by inactivating myeloid suppressor cells

This is the author's accepted manuscript. The final published article is available from the link below. Note: In this manuscript as well as in the original published version of this article the word "Polyphenon" was incorrectly spelled in the title as "Polyphenol."Purpose: Neuroblastoma is a rare childhood cancer whose high risk, metastatic form has a dismal outcome in spite of aggressive therapeutic interventions. The toxicity of drug treatments is a major problem in this pediatric setting. In this study, we investigated whether Polyphenon E, a clinical grade mixture of green tea catechins under evaluation in multiple clinical cancer trials run by the National Cancer Institute (Bethesda, MD), has anticancer activity in mouse models of neuroblastoma. Experimental Design: We used three neuroblastoma models: (i) transgenic TH-MYCN mouse developing spontaneous neuroblastomas; (ii) nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenotransplanted with human SHSY5Y cells; and (iii) A/J mice transplanted with syngeneic Neuro 2A cells. Mice were randomized in control and Polyphenon E–drinking groups. Blood from patients with neuroblastoma and normal controls was used to assess the phenotype and function of myeloid cells. Results: Polyphenon E reduced the number of tumor-infiltrating myeloid cells, and inhibited the development of spontaneous neuroblastomas in TH-MYCN transgenic mice. In therapeutic models of neuroblastoma in A/J, but not in immunodeficient NOD/SCID mice, Polyphenon E inhibited tumor growth by acting on myeloid-derived suppressor cells (MDSC) and CD8 T cells. In vitro, Polyphenon E impaired the development and motility of MDSCs and promoted differentiation to more neutrophilic forms through the 67 kDa laminin receptor signaling and induction of granulocyte colony-stimulating factor. The proliferation of T cells infiltrating a patient metastasis was reactivated by Polyphenon E. Conclusions: These findings suggest that the neuroblastoma-promoting activity of MDSCs can be manipulated pharmacologically in vivo and that green tea catechins operate, at least in part, through this mechanism.SPARKS, Research in Childhood Cancer, the CGD Research Trust, and the Wellcome Trust

Estimating the prevalence of latent tuberculosis in a low-incidence setting: Australia.

Migration is a key driver of tuberculosis (TB) in many low-incidence settings, with the majority of TB cases attributed to reactivation of latent TB (LTBI) acquired overseas. A greater understanding of LTBI risk in heterogeneous migrant populations would aid health planning. We aimed to estimate the LTBI prevalence and distribution among locally born and overseas-born Australians.Annual risks of TB infection estimates were applied to population cohorts (by country of birth, year of arrival and age) in Australian census data in 2006, 2011 and 2016.Both the absolute number and proportion of Australian residents with LTBI increased from 4.6% (interquartile range (IQR) 4.2-5.2%) in 2006 to 5.1% (IQR 4.7-5.5%) in 2016, due to the increasing proportion of the population born overseas (23.8% in 2006 to 28.3% in 2016). Of all residents estimated to have LTBI in 2016; 93.2% were overseas born, 21.6% were aged <35 years and 34.4% had migrated to Australia since 2007.The overall prevalence of LTBI in Australia is low. Some residents, particularly migrants from high-incidence settings, may have considerably higher risk of LTBI, and these findings allow for tailored public health interventions to reduce the risk and impact of future TB disease

The timing of auditory sensory deficits in Norrie disease has implications for therapeutic intervention

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease

Hormone Therapy and Young-Onset Breast Cancer

Estrogen plus progestin hormone therapy (HT) is associated with an increased risk of postmenopausal breast cancer, but few studies have examined the impact of HT use on the risk of breast cancer in younger women. We assessed the association between estrogen plus progestin HT or unopposed estrogen HT and young-onset breast cancer using data from the Two Sister Study (2008–2010), a sister-matched study of 1,419 cases diagnosed with breast cancer before the age of 50 years and 1,665 controls. We assessed exposures up to a family-specific index age to ensure comparable opportunities for exposures and used propensity scores to control for birth cohort effects on HT use. Ever HT use was uncommon (7% and 11% in cases and controls, respectively). Use of estrogen plus progestin was not associated with an increased risk of young-onset breast cancer (odds ratio = 0.80, 95% confidence interval: 0.41, 1.59). Unopposed estrogen use was inversely associated with the risk of young-onset breast cancer (odds ratio = 0.58, 95% confidence interval: 0.34, 0.99). Duration of use, age at first use, and recency of use did not modify these associations

Impact of Reversion of Mycobacterium tuberculosis Immunoreactivity Tests on the Estimated Annual Risk of Infection

A key metric in tuberculosis epidemiology is the annual risk of infection (ARI), which is usually derived from tuberculin skin test (TST) and interferon-gamma release assay (IGRA) prevalence surveys in children. Deriving the ARI assumes that immunoreactivity is persistent over time; however, reversion of immunoreactivity has long been documented. Here we use a deterministic, compartmental model of Mycobacterium tuberculosis (Mtb) infection to explore the impact of reversion on ARI estimation using age-specific reversion probabilities for TST and IGRA. Using empirical data of TST reversion (22.2%/year for 0-19yo), the true ARI is 2-5 times higher than estimated from immunoreactivity studies in 8-12 year-olds. Applying empirical reversion probabilities for IGRA (9.9%/year for 12-18yo) showed a 1.5-2-fold underestimation. ARIs are increasingly underestimated in older populations, due to the cumulative impact of reversion on population reactivity over time. Declines in annual risk did not largely affect the results. Ignoring reversion leads to a stark underestimation of the true ARI in populations and our interpretation of Mtb transmission intensity. Future surveys should adjust for reversion probabilities and its cumulative effect with increasing age to provide a more accurate reflection of the burden and dynamics of Mtb infection