Abstract High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.
Introduction: The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by activation of NF-kB signaling and an increased risk of mortality. Recurrent somatic mutations affecting genes such as MYD88, CD79A/B and TNFAIP3 have been shown to constitutively activate the NF-kB pathway through B-cell receptor signaling in ABC DLBCL; however, there still remain cases with no known genetic basis for this pathway activation (Arthur et al. Nat Com 2018). We recently published a meta-analysis of DLBCL genome and targeted sequencing data identifying non-coding mutations. We described novel mutations affection the 3′ untranslated region (UTR) of NFKBIZ in 18% of ABC DLBCLs. Overall, NFKBIZ is mutated (amplifications and UTR mutations) in 34% of ABC DLBCLs. These NFKBIZ mutations are mutually exclusive with MYD88 mutations, implicating them in activation of the NF-kB signaling pathway. NFKBIZ encodes the IkB-ζ protein, which interacts with NF-kB transcription factors and is thought to regulate canonical NF-kB signaling. We hypothesized that these mutations affect the ability of regulatory mechanisms to target this transcript for degradation through disruption of UTR secondary structures. This leads to enhanced mRNA stability and elevated protein levels and represents a novel mechanism of promoting NF-kB signaling in ABC DLBCL. Methods: NFKBIZ 3′ UTR mutations were introduced in a DLBCL cell line using CRISPR-Cas9. NFKBIZ mRNA and protein levels were evaluated using custom designed droplet digital PCR assays and western blot. RNA-sequencing was performed on mutant and wild-type (WT) cell lines to identify genes up-regulated by IkB-ζ. A competitive growth assay with WT and CRISPR mutant lines was set up to assess whether UTR mutations provide a growth advantage in culture. The pool composition was determined by DNA sequencing and comparison of WT and mutant DNA sequences. Results: Introduction of NFKBIZ mutations into DLBCL cell lines confirmed that UTR deletions lead to increased levels of mRNA and protein. Stimulation with LPS revealed that mRNA levels stay elevated for longer in mutant lines. NFKBIZ UTR deletions also give DLBCL cells a selective growth advantage over WT when grown together in culture. RNA-sequencing of mutant and WT lines revealed possible transcriptional targets of IkB-ζ, including TNFRSF14B, HCK, GNAZ, BATF and CD274. These targets are either involved in activation of NF-kB signaling, associated with decreased survival in other lymphomas or potential new drug targets in NFKBIZ mutant DLBCL. Conclusions: This work highlights the role of NFKBIZ and 3′ UTR mutations in driving ABC DLBCL. We demonstrate that these UTR mutations can lead to over-expression of NFKBIZ and activate potentially novel drug targets in ABC DLBCL. These findings contribute to a better understanding of the genetic basis of DLBCL, which is necessary to guide personalized therapeutic strategies. Keywords: activated B-cell-like (ABC); molecular genetics; NF-kB.
<div>Abstract<p>Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but extremely lethal malignancy that mainly impacts young women. SCCOHT is characterized by a diploid genome with loss of SMARCA4 and lack of SMARCA2 expression, two mutually exclusive ATPases of the SWI/SNF chromatin-remodeling complex. We and others have identified the histone methyltransferase EZH2 as a promising therapeutic target for SCCOHT, suggesting that SCCOHT cells depend on the alternation of epigenetic pathways for survival. In this study, we found that SCCOHT cells were more sensitive to pan-HDAC inhibitors compared with other ovarian cancer lines or immortalized cell lines tested. Pan-HDAC inhibitors, such as quisinostat, reversed the expression of a group of proteins that were deregulated in SCCOHT cells due to SMARCA4 loss, leading to growth arrest, apoptosis, and differentiation <i>in vitro</i> and suppressed tumor growth of xenografted tumors of SCCOHT cells. Moreover, combined treatment of HDAC inhibitors and EZH2 inhibitors at sublethal doses synergistically induced histone H3K27 acetylation and target gene expression, leading to rapid induction of apoptosis and growth suppression of SCCOHT cells and xenografted tumors. Therefore, our preclinical study highlighted the therapeutic potential of combined treatment of HDAC inhibitors with EZH2 catalytic inhibitors to treat SCCOHT.</p></div>
Abstract Natural Killer (NK) lymphocytes are innate immune cells, mainly known to eliminate cancerous and virus infected cells. The NCR1/NKp46 receptor is expressed specifically in the NK lineage. Activation of NK effector functions results when the receptor binds viral haemagglutinin or unidentified tumor ligands. Knockout studies have shown that the molecule is indispensible in the control of influenza infection, lymphoma growth, and the development of type I diabetes. Yet the molecular basis behind the specificity of NCR1 expression is unknown. We seek to uncover the genetic and epigenetic mechanisms that regulate human NCR1 transcription. Comparative genomics and transient luciferase assays suggest that crucial cis-regulatory elements are found within 300bp upstream of the gene. Within this proximal region, luciferase experiments also indicate the presence of a basal, non-specific promoter as well as a tissue-specific regulatory switch. Through bioinformatics and transcription factor RNA expression screens, RUNX3 was identified as a probable regulator of the switch. Chromatin immunoprecipitation (ChIP) studies were conducted to confirm RUNX3 binding in NK cells. A proteomics approach is currently underway to identify additional factors. Finally, preliminary ChIP data hints at transcriptional control by histone 3 lysine 4 methylation in blood lineages.
