Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/β-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/β-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/β-catenin signaling is activated in ovarian CICs, and targeted inhibition of β-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/β-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/β-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option.

SummaryTelomerase is a reverse transcriptase specialized in telomere synthesis. The enzyme is primarily nuclear where it elongates telomeres, but many reports show that the catalytic component of telomerase (in humans called hTERT) also localizes outside of the nucleus, including in mitochondria. Shuttling of hTERT between nucleus and cytoplasm and vice versa has been reported, and different proteins shown to regulate such translocation. Exactly why telomerase moves between subcellular compartments is still unclear. In this study we report that mutations that disrupt the nuclear export signal (NES) of hTERT render it nuclear but unable to immortalize cells despite retention of catalytic activity in vitro. Overexpression of the mutant protein in primary fibroblasts is associated with telomere-based cellular senescence, multinucleated cells and the activation of the DNA damage response genes ATM, Chk2 and p53. Mitochondria function is also impaired in the cells. We find that cells expressing the mutant hTERT produce high levels of mitochondrial reactive oxygen species and have damage in telomeric and extratelomeric DNA. Dysfunctional mitochondria are also observed in an ALT (alternative lengthening of telomeres) cell line that is insensitive to growth arrest induced by the mutant hTERT showing that mitochondrial impairment is not a consequence of the growth arrest. Our data indicate that mutations involving the NES of hTERT are associated with defects in telomere maintenance, mitochondrial function and cellular growth, and suggest targeting this region of hTERT as a potential new strategy for cancer treatment.

Background Remdesivir is efficacious for severe COVID-19 in adults, but data in pregnant women are limited. We describe outcomes in the first 86 pregnant women with severe COVID-19 who were treated with remdesivir. Methods Reported data span March 21 to June 16, 2020 for hospitalized pregnant women with PCR-confirmed SARS-CoV-2 infection and room air oxygen saturation ≤94% whose clinicians requested remdesivir through the compassionate use program. The intended remdesivir treatment course was 10 days (200mg on Day 1, followed by 100mg for Days 2-10, given intravenously). Results Nineteen of 86 women delivered before their first dose and were reclassified as immediate “postpartum” (median postpartum day=1; range 0-3). At baseline, 40% of pregnant women (median gestational age 28 weeks) required invasive ventilation, in contrast to 95% of postpartum women (median gestational age at delivery 30 weeks). By Day 28 of follow-up, the level of oxygen requirement decreased in 96% and 89% of pregnant and postpartum women, respectively. Among pregnant women, 93% of those on mechanical ventilation were extubated, 93% recovered, and 90% were discharged. Among postpartum women, 89% were extubated, 89% recovered, and 84% were discharged. Remdesivir was well tolerated, with a low incidence of serious adverse events (16%). Most adverse events were related to pregnancy and underlying disease; most laboratory abnormalities were Grades 1 or 2. There was one maternal death attributed to underlying disease and no neonatal deaths. Conclusions Among 86 pregnant and postpartum women with severe COVID-19 who received compassionate use remdesivir, recovery rates were high, with a low rate of serious adverse events.

Ataxia telangiectasia (A-T) is a progressive childhood disorder characterized most notably by cerebellar degeneration and predisposition to cancer. A-T is caused by mutations in the kinase ATM, a master regulator of the DNA double-strand break response. In addition to DNA-damage signaling defects, A-T cells display mitochondrial dysfunction that is thought to contribute to A-T pathogenesis. However, the molecular mechanism leading to mitochondrial dysfunction in A-T remains unclear. Here we show that lack of ATM leads to reduced mitochondrial DNA (mtDNA) integrity and mitochondrial dysfunction, which are associated to defective mtDNA repair. While protein levels of mtDNA repair proteins are essentially normal, in the absence of ATM levels specifically of DNA ligase III (Lig3), the only DNA ligase working in mitochondria are reduced. The reduction of Lig3 is observed in different A-T patient cells, in brain and pre-B cells derived from ATM knockout mice as well as upon transient or stable knockdown of ATM. Furthermore, pharmacological inhibition of Lig3 in wild type cells phenocopies the mtDNA repair defects observed in A-T patient cells. As targeted deletion of LIG3 in the central nervous system causes debilitating ataxia in mice, reduced Lig3 protein levels and the consequent mtDNA repair defect may contribute to A-T neurodegeneration. A-T is thus the first disease characterized by diminished Lig3.

This unit describes the gene-specific quantitative PCR-based (QPCR) assay, which is used to measure DNA integrity of both nuclear and mitochondrial genomes based on amplification of long DNA targets. QPCR can be used to quantify the formation of DNA damage and the kinetics of DNA repair by following restoration of amplification of the target DNA over time after removal of the damaging agent. A detailed protocol to set up QPCR in any laboratory, highlighting critical parameters for successful establishment of the assay and interpretation of the results, is provided here. Advantages (e.g., the use of nanogram amounts of DNA) and limitations (e.g., the inability to define the specific type of lesion present on the DNA) of using QPCR to assay DNA damage in human cells are also described.

Computation-based drug-repurposing/repositioning approaches can greatly speed up the traditional drug discovery process. To date, systematic and comprehensive computation-based approaches to identify and validate drug-repositioning candidates for epithelial ovarian cancer (EOC) have not been undertaken. Here, we present a novel drug discovery strategy that combines a computational drug-repositioning system (DrugPredict) with biological testing in cell lines in order to rapidly identify novel drug candidates for EOC. DrugPredict exploited unique repositioning opportunities rendered by a vast amount of disease genomics, phenomics, drug treatment, and genetic pathway and uniquely revealed that non-steroidal anti-inflammatories (NSAIDs) rank just as high as currently used ovarian cancer drugs. As epidemiological studies have reported decreased incidence of ovarian cancer associated with regular intake of NSAIDs, we assessed whether NSAIDs could have chemoadjuvant applications in EOC and found that (i) NSAID Indomethacin induces robust cell death in primary patient-derived platinum-sensitive and platinum- resistant ovarian cancer cells and ovarian cancer stem cells and (ii) downregulation of β-catenin is partially driving effects of Indomethacin in cisplatin-resistant cells. In summary, we demonstrate that DrugPredict represents an innovative computational drug- discovery strategy to uncover drugs that are routinely used for other indications that could be effective in treating various cancers, thus introducing a potentially rapid and cost-effective translational opportunity. As NSAIDs are already in routine use in gynecological treatment regimens and have acceptable safety profile, our results will provide with a rationale for testing NSAIDs as potential chemoadjuvants in EOC patient trials.

Decreased placental oxygenation and increased oxidative stress are implicated in the development of preeclampsia. Oxidative stress arises from imbalance between pro-versus anti-oxidants and can lead to biological oxidation and apoptosis. Because pregnant women living at high altitude (3100 m, HA) have lowered arterial PO2 and an increased incidence of preeclampsia, we hypothesized that HA placentas would have decreased anti-oxidant enzyme activity, increased oxidative stress (lipid peroxidation, protein oxidation and nitration) and greater trophoblast apoptosis than low-altitude (LA) placentas. We measured enzymatic activities, lipid and protein oxidation and co-factor concentrations by spectrophotometric techniques and ELISA in 12 LA and 18 HA placentas. Immunohistochemistry (IHC) was used to evaluate nitrated proteins and specific markers of apoptosis (activated caspase 3 and M30). Superoxide dismutase activity was marginally lower (p=0.05), while glutathione peroxidase activity (p<0.05), thioredoxin concentrations (p<0.005) and thioredoxin reductase activity p<0.01 were all reduced in HA placentas. Decreased anti-oxidant activity was not associated with increased oxidative stress: lipid peroxide content and protein carbonyl formation were lower at HA (p<0.01). We found greater nitrotyrosine residues in the syncytiotrophoblast at 3100 m (p<0.05), but apoptosis did not differ between altitudes. Our data suggest that hypoxia does not increase placental oxidative stress in vivo. Nitrative stress may be a consequence of hypoxia but does not appear to contribute to increased apoptosis. Lowered placental concentrations of anti-oxidants may contribute to the susceptibility of women living at HA to the development of preeclampsia, but are unlikely to be etiological.

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-⌬T or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-⌬T have normal dynactin "comets" at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types. INTRODUCTIONThe radial array of microtubules is typical for many mammalian cells. It organizes bidirectional organelle transport in the cytoplasm in the endocytotic and exocytotic direction. It is also required for the regulation of interaction of microtubule plus ends with cell periphery. Both functions are important for cell polarization, movement, and signal transduction (Hyman and Karsenti, 1996;Dujardin et al., 2003;Morrison, 2007). The degree of radiality of microtubules varies in different types of cells. For instance, in fish melanophores the system of microtubules is perfectly radial (Schliwa et al., 1978), whereas in myotubes it is unclear (Tassin et al., 1985;Musa et al., 2003). Moreover, among fibroblast-like cultured mammalian cells some (green monkey kidney Vero or Chinese hamster ovary CHO-K1) possess a distinct radial microtubule array (Bre et al., 1987), whereas others (human cervical carcinoma HeLa or mouse fibroblasts NIH 3T3) have rather chaotic microtubule arrangements (Bulinski and Borisy, 1980). Regardless of the tissue origin of cells, microtubules become more radial when cultured cells are sparse and less radial in confluent cultures. It seems that the status of microtubule organization is regulated by signal transduction pathways and depends on cell differentiation.The focused microtubule arrays can also be formed in acentrosomal cell fragments as a result of interactions between microtubules and membrane vesicles, covered with motor proteins (Rodionov and Borisy, 1997;Malikov et al., 2005). In acentrosomal fragments of fish melanocytes microtubules are chaotic when pigment granules are dispersed. After the addition of adrenaline or other activation of melanosome aggregation microtubules assemble in a radial array. Thus, in this case...