Detection of SARS-CoV-2 RNA using RT-LAMP and molecular beacons.

BACKGROUND: Rapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse transcription-polymerase chain reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but instrument costs are high and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse transcription and loop-mediated isothermal amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. RESULTS: Here we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. CONCLUSIONS: LAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.

MMB-FOXM1-driven premature mitosis is required for CHK1 inhibitor sensitivity.

To identify genes whose loss confers resistance to CHK1 inhibitors, we perform genome-wide CRISPR-Cas9 screens in non-small-cell lung cancer (NSCLC) cell lines treated with the CHK1 inhibitor prexasertib (CHK1i). Five of the top six hits of the screens, MYBL2 (B-MYB), LIN54, FOXM1, cyclin A2 (CCNA2), and CDC25B, are cell-cycle-regulated genes that contribute to entry into mitosis. Knockout of MMB-FOXM1 complex components LIN54 and FOXM1 reduce CHK1i-induced DNA replication stress markers and premature mitosis during Late S phase. Activation of a feedback loop between the MMB-FOXM1 complex and CDK1 is required for CHK1i-induced premature mitosis in Late S phase and subsequent replication catastrophe, indicating that dysregulation of the S to M transition is necessary for CHK1 inhibitor sensitivity. These findings provide mechanistic insights into small molecule inhibitors currently studied in clinical trials and provide rationale for combination therapies.

ALC1 links chromatin accessibility to PARP inhibitor response in homologous recombination-deficient cells.

The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.

CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress.

While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.

NF-κB inhibition by dimethylaminoparthenolide radiosensitizes non-small-cell lung carcinoma by blocking DNA double-strand break repair.

Despite optimal chemotherapy, radiotherapy (RT), and/or surgery, non-small-cell lung carcinoma (NSCLC) remains the leading cause of cancer-related death in the US and worldwide. Thoracic RT, a mainstay in the treatment of locally advanced NSCLC, is often restricted in efficacy by a therapeutic index limited by sensitivity of tissues surrounding the malignancy. Therefore, radiosensitizers that can improve the therapeutic index are a vital unmet need. Inhibition of the NF-κB pathway is a proposed mechanism of radiosensitization. Here we demonstrate that inhibition of the canonical NF-κB pathway by dimethylaminoparthenolide (DMAPT) radiosensitizes NSCLC by blocking DNA double-strand break (DSB) repair. NF-κB inhibition results in significant impairment of both homologous recombination (HR) and non-homologous end joining (NHEJ), as well as reductions in ionizing radiation (IR)-induced DNA repair biomarkers. NF-κB inhibition by DMAPT shows preclinical potential for further investigation as a NSCLC radiosensitizer.

Circulating miR-29a and miR-150 correlate with delivered dose during thoracic radiation therapy for non-small cell lung cancer.

BACKGROUND: Risk of normal tissue toxicity limits the amount of thoracic radiation therapy (RT) that can be routinely prescribed to treat non-small cell lung cancer (NSCLC). An early biomarker of response to thoracic RT may provide a way to predict eventual toxicities-such as radiation pneumonitis-during treatment, thereby enabling dose adjustment before the symptomatic onset of late effects. MicroRNAs (miRNAs) were studied as potential serological biomarkers for thoracic RT. As a first step, we sought to identify miRNAs that correlate with delivered dose and standard dosimetric factors. METHODS: We performed miRNA profiling of plasma samples obtained from five patients with Stage IIIA NSCLC at five dose-points each during radical thoracic RT. Candidate miRNAs were then assessed in samples from a separate cohort of 21 NSCLC patients receiving radical thoracic RT. To identify a cellular source of circulating miRNAs, we quantified in vitro miRNA expression intracellularly and within secreted exosomes in five NSCLC and stromal cell lines. RESULTS: miRNA profiling of the discovery cohort identified ten circulating miRNAs that correlated with delivered RT dose as well as other dosimetric parameters such as lung V20. In the validation cohort, miR-29a-3p and miR-150-5p were reproducibly shown to decrease with increasing radiation dose. Expression of miR-29a-3p and miR-150-5p in secreted exosomes decreased with radiation. This was concomitant with an increase in intracellular levels, suggesting that exosomal export of these miRNAs may be downregulated in both NSCLC and stromal cells in response to radiation. CONCLUSIONS: miR-29a-3p and miR-150-5p were identified as circulating biomarkers that correlated with delivered RT dose. miR-150 has been reported to decrease in the circulation of mammals exposed to radiation while miR-29a has been associated with fibrosis in the human heart, lungs, and kidneys. One may therefore hypothesize that outlier levels of circulating miR-29a-3p and miR-150-5p may eventually help predict unexpected responses to radiation therapy, such as toxicity.

Efficacy of Analgesic Treatments to Manage Children's Postoperative Pain After Laparoscopic Appendectomy: Retrospective Medical Record Review.

Knowledge of the effectiveness of multimodal analgesic treatments to manage children's postoperative pain during hospital stays is limited. Our retrospective chart review of a convenience sample of 200 pediatric surgical patients' pain experiences during the first 24 hours after laparoscopic appendectomy demonstrates the benefits of a multimodal analgesic approach. We found that pediatric patients who received perioperative IV ketorolac in addition to opioids reported statistically significantly lower mean pain intensity (n = 134, mean [M] = 2.9, standard deviation [SD] = 1.7) during the first 24 hours after surgery when compared with the pain intensity of patients who did not receive perioperative IV ketorolac (n = 66, M = 3.7, SD = 1.7, t = 3.14, P = .002). Patients who received perioperative IV ketorolac (M = 0.94, SD = 0.71) also received significantly fewer morphine equivalents of postoperative opioids during the first 24 hours after surgery than those who did not (M = 1.21, SD = 0.78, t = 2.41, P = .02). We will use data from these patients to introduce the potential for a personalized medicine approach to postoperative pain.

USP9X inhibition promotes radiation-induced apoptosis in non-small cell lung cancer cells expressing mid-to-high MCL1.

BACKGROUND AND PURPOSE: Radiotherapy (RT) is vital for the treatment of locally advanced non-small cell lung cancer (NSCLC), yet its delivery is limited by tolerances of adjacent organs. We sought therefore to identify and characterize gene targets whose inhibition may improve RT. MATERIALS AND METHODS: Whole genome pooled shRNA cytotoxicity screens were performed in A549 and NCI-H460 using a retroviral library of 74,705 sequences. Cells were propagated with or without daily radiation Monday-Friday. Radiosensitization by top differential dropout hits was assessed by clonogenic assays. Apoptosis was assessed using a caspase 3/7 cell-based activity assay and by annexin V-FITC and PI staining. MCL1 expression was assessed by qPCR and Western blotting. RESULTS: USP9X, a deubiquitinase, was a top hit among druggable gene products. WP1130, a small molecule USP9X inhibitor, showed synergistic cytotoxicity with IR. MCL1, an anti-apoptotic protein deubiquitinated by USP9X, decreased with USP9X inhibition and IR. This was accompanied by increases in caspase 3/7 activity and apoptosis. In a panel of NSCLC lines, MCL1 and USP9X protein and gene expression levels were highly correlated. Lines showing high levels of MCL1 expression were the most sensitive to USP9X inhibition. CONCLUSIONS: These data support the use of MCL1 expression as a predictive biomarker for USP9X inhibitors in NSCLC therapy.