Development and Clinical Evaluation of a CRISPR/Cas12a-Based Nucleic Acid Detection Platform for the Diagnosis of Keratomycoses.

Objective: The objective of this study was to develop a rapid and accurate clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based molecular diagnostic assay (Rapid Identification of Mycoses using CRISPR, RID-MyC assay) to detect fungal nucleic acids and to compare it with existing conventional mycologic methods for the diagnosis of fungal keratitis (FK). Design: This study was structured as a development and validation study focusing on the creation and assessment of the RID-MyC assay as a novel diagnostic modality for FK. Subjects: Participants comprised 142 individuals presenting with suspected microbial keratitis at 3 tertiary care institutions in South India. Methods: The RID-MyC assay utilized recombinase polymerase amplification targeting the 18S ribosomal RNA gene for isothermal amplification, followed by a CRISPR/Cas12a reaction. This was benchmarked against microscopy, culture, and polymerase chain reaction for the diagnosis of FK. Main Outcome Measures: The primary outcome measures focused on the analytical sensitivity and specificity of the RID-MyC assay in detecting fungal nucleic acids. Secondary outcomes measured the assay's diagnostic sensitivity and specificity for FK, including its concordance with conventional diagnostic methods. Results: The RID-MyC assay exhibited a detection limit ranging from 13.3 to 16.6 genomic copies across 4 common fungal species. In patients with microbial keratitis, the RID-MyC assay showed substantial agreement with microscopy (kappa = 0.714) and fair agreement with culture (kappa = 0.399). The assay demonstrated a sensitivity of 93.27% (95% confidence interval [CI], 86.62%-97.25%) and a specificity of 89.47% (95% CI, 66.86%-98.70%) for FK diagnosis, with a median diagnostic time of 50 minutes (range, 35-124 minutes). Conclusions: The RID-MyC assay, utilizing CRISPR-Cas12a technology, offers high diagnostic accuracy for FK. Its potential for point-of-care use could expedite and enhance the precision of fungal diagnostics, presenting a promising solution to current diagnostic challenges. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

OCT4-mediated transcription confers oncogenic advantage for a subset of gastric tumors with poor clinical outcome.

Deregulated transcription programs and signaling pathways are the critical factors involved in the process of carcinogenesis. Signaling pathway-based classification of tumors is expected to pave the way for the development of targeted therapeutics. We investigated the OCT4-mediated transcription program in the gene expression profiles of 939 gastric tumor samples. A set of 84 genes showing positive correlation with the activation pattern of the available OCT4 gene sets were found to consistently express in diffuse, poorly differentiated, and stage-III gastric tumors with poor prognosis. We also developed stable OCT4-silenced gastric cancer cells and the resultant gene expression changes were investigated by genome-wide mRNA profiling. Functional genomic investigation of the genes downregulated in OCT4-silenced cells and the pathways co-activated with OCT4 gene set across gastric tumors revealed the positive association of dysregulated OCT4 with TGF-ß, GLI, PRC2/EzH2, Wnt, KRAS, STK33, and YAP signaling pathways in diffuse subtype gastric tumors. Elevated expression of OCT4 gene set was identified to represent the previously described EMT_UP as well as the GENOMICALLY STABLE subtypes of gastric tumors. Integrative genomic screening of the drug sensitivity of gastric cancer cells in correlation with the expression of OCT4 gene set across drug sensitivity databases revealed the inhibitors of tyrosine kinases, HDAC, and HSP90 to have a negative correlation and needs to be investigated for their potential therapeutic features for the subset of OCT4-activated gastric tumors. Thus, the subset of gastric tumors with OCT4 activation, the associated oncogenic signaling pathways, and potential therapeutic candidates were identified for the development of targeted therapeutic strategies.

Delineation of gastric tumors with activated ERK/MAPK signaling cascades for the development of targeted therapeutics.

The ERK/MAPK signaling pathway is activated in various cancers including gastric cancer. Targeting the ERK/MAPK/MEK pathway has been considered as a promising strategy for cancer therapy. However, MEK inhibition leads to a series of resistance mechanisms due to mutations in MEK, elevated expression of RAS or RAF proteins and activation of the associated signaling pathways. In the present study, ERK/MAPK pathway specific gene signatures were identified to be highly activated in intestinal subtype gastric tumors. Inhibition of ERK/MAPK pathway with the inhibitor PD98059 in gastric cancer cell lines by in vitro signaling pathway and genome-wide expression profiling revealed the associated signaling pathways. Functional genomic investigation of the ERK/MAPK regulated genes reveals the association of ERK/MAPK pathway with E2F, Myc, SOX-2, TGF-ß, OCT4 and Notch pathways in gastric cancer cells. Of these, E2F, Myc and SOX-2 pathways are activated in intestinal subtype gastric tumors and TGF-ß, OCT4, Notch pathways are activated in diffuse subtype gastric tumors. Further, the mutational load of gastric tumors was found to have association and correlation with the activation pattern of ERK/MAPK pathways across gastric tumors. ERK/MAPK activation was also found to represent the EBV and MSI activated subtypes of gastric tumors. Identification of potent drug candidates inhibiting the ERK/MAPK and associated pathways would pave a way for developing the targeted therapeutics for a subset of gastric tumors with activated ERK/MAPK signaling cascade.

Small peptide inhibitor from the sequence of RUNX3 disrupts PAK1-RUNX3 interaction and abrogates its phosphorylation-dependent oncogenic function.

P21 Activated Kinase 1 (PAK1) is an oncogenic serine/threonine kinase known to play a significant role in the regulation of cytoskeleton and cell morphology. Runt-related transcription factor 3 (RUNX3) was initially known for its tumor suppressor function, but recent studies have reported the oncogenic role of RUNX3 in various cancers. Previous findings from our laboratory provided evidence that Threonine 209 phosphorylation of RUNX3 acts as a molecular switch in dictating the tissue-specific dualistic functions of RUNX3 for the first time. Based on these proofs and to explore the translational significance of these findings, we designed a small peptide (RMR) from the protein sequence of RUNX3 flanking the Threonine 209 phosphorylation site. The selection of this specific peptide from multiple possible peptides was based on their binding energies, hydrogen bonding, docking efficiency with the active site of PAK1 and their ability to displace PAK1-RUNX3 interaction in our prediction models. We found that this peptide is stable both in in vitro and in vivo conditions, not toxic to normal cells and inhibits the Threonine 209 phosphorylation in RUNX3 by PAK1. We also tested the efficacy of this peptide to block the RUNX3 Threonine 209 phosphorylation mediated tumorigenic functions in in vitro cell culture models, patient-derived explant (PDE) models and in in vivo tumor xenograft models. These results proved that this peptide has the potential to be developed as an efficient therapeutic molecule for targeting RUNX3 Threonine 209 phosphorylation-dependent tumor phenotypes.

Identification of the targeted therapeutic potential of doxycycline for a subset of gastric cancer patients.

The identification of new drugs for the targeted therapy of gastric cancer remains an important need. The RAS/RAF/MEK/ERK/ELK1 signaling cascade is activated in many cancers, including gastric cancer. To identify the targetable inhibitors of the ERK/MAPK pathway, we performed a repurposing screening of a panel of antimicrobial agents in gastric cancer cells using an ERK/MAPK-driven firefly luciferase reporter assay. Multiple antibiotics were identified to inhibit ERK-mediated transcriptional activity. Among them, doxycycline showed high inhibition of ERK/MAPK-regulated transcriptional activity and the levels of ERK proteins. Doxycycline was further identified to inhibit the proliferation and the colony- and spheroid-forming potential of gastric cancer cells. By in vitro signaling pathway and genome-wide expression profiling analyses, doxycycline was identified to inhibit signaling pathways and transcriptional activities regulated by ER, Myc, E2F1, Wnt, SMAD2/3/4, Notch, and OCT4. Doxycycline was also found to activate p53-, ATF6-, NRF1/2-, and MTF1-mediated transcription and inhibit the transcription of histones, proteasomal genes, fibroblast growth factor, and other oncogenic factors. These observations show the multitargeting and targeted therapeutic features of doxycycline for a subset of gastric tumors.

NFκB activation demarcates a subset of hepatocellular carcinoma patients for targeted therapy.

BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer death worldwide. It is a heterogeneous disorder and >80 % of the tumors develop in patients with liver cirrhosis, resulting from chronic inflammation and/or fibrosis. Here, we set out to identify novel targets for HCC therapy and to define a subgroup of patients that might benefit most from it. METHODS: Cellular pathway activation profiling of 45 transcription factors in a HCC-derived cell line (HEP3B), in vitro analysis of NFκB reporter activity in additional HCC-derived cell lines and pathway-focused integrative analyses of publicly available primary HCC-derived expression profiling data (GSE6764, GSE9843, E-TABM-36 and E-TABM-292) were employed to reveal a role of NFκB in HCC development. In order to identify potential targeting agents, a luciferase-based NFκB reporter screening assay was established in HEP3B cells. After screening of a drug library through this assay, a potent NFκB pathway inhibitor was identified and characterized using an array of additional in vitro assays. RESULTS: Using cellular pathway activation profiling, we found a high activation of NFκB-mediated signaling in HCC-derived cell lines and in primary HCC tumors. Through NFκB inhibitor screening we observed a highly efficacious NFκB pathway inhibitory potential of ornithogalum in HCC-derived HEP3B cells. Although its active component still remains to be defined, ornithogalum has been found to inhibit endoplasmic reticulum (ER) and oxidative stress responses. ER stress, oxidative stress and NFκB signaling were found to be enhanced in a subset of HCCs, as well as in (precancerous) liver cirrhosis tissues. CONCLUSION: From our data we conclude that NFκB signaling is activated in precancerous cirrhosis tissues and in a subset of HCCs. We found that ornithogalum exhibits NFκB targeting and stress relieving activities. NFκB inhibitors, including the active component of ornithogalum, may serve as putative preventive and targeted therapeutic agents for at least a subset of HCCs in which the NFκB pathway is activated. These latter notions require further investigation in a translational context.