NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer.

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The proapoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models, we identified an inhibitor of the transcription factor heterodimer CBFß/RUNX1. By genetic gain and loss of function experiments, we validated that the mode of action depends on RUNX1 and NOXA. Of note is that RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome-wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a way to target a therapy-resistant PDAC, an unmet clinical need.

Can energy drinks affect the surface quality of bioactive restorative materials?

BACKGROUNDS: This study aimed to compare the effects of different energy drinks on the surface roughness, weight loss, and color change of various bioactive restorative materials. METHODS: Charisma Diamond One, Activa™ BioActive Restorative, Activa™ Presto™ and Equia Forte HT Fil samples were prepared using plastic molds (8 × 2 mm) (n = 10/groups). After polishing, the samples were weighed, their colors were recorded using a spectrophotometer according to the CIEDE2000 system, and their surface roughness was measured using a profilometer. The samples were immersed in Powerade, Burn, Monster and distilled water for 7 days. After immersion, all the measurements were repeated. Statistical analyses were performed using the Wilcoxon signed-rank test and the Mann‒Whitney U test (p < 0.05). RESULTS: All energy drinks roughened the surface of Equia Forte HT Fil (p < 0.05). Powerade and Monster increased the Ra of all materials after 7 days (p < 0.05). Burns affected all materials except the Activa Bioactive (p < 0.05). Significant weight loss was observed in the Equia Forte group after immersion in all the energy drinks, whereas no weight loss was observed in the other groups. According to the color measurements, ΔE00 values were greater in the Burn and Monster groups, except for the Equia Forte HT Fil group (p < 0.05). CONCLUSION: Energy drinks affected bioactive materials to varying degrees. The glass hybrid material was the most affected, and the bioactive restorative materials based on the resin matrix were the least.

Small-molecule SUMO inhibition for biomarker-informed B-cell lymphoma therapy.

Aberrant activity of the SUMOylation pathway has been associated with MYC overexpression and poor prognosis in aggressive B-cell lymphoma (BCL) and other malignancies. Recently developed small-molecule inhibitors of SUMOylation (SUMOi) target the heterodimeric E1 SUMO activation complex (SAE1/UBA2). Here, we report that activated MYC signaling is an actionable molecular vulnerability in vitro and in a preclinical murine in vivo model of MYC-driven BCL. While SUMOi conferred direct effects on MYC-driven lymphoma cells, SUMO inhibition also resulted in substantial remodeling of various subsets of the innate and specific immunity in vivo. Specifically, SUMOi increased the number of memory B cells as well as cytotoxic and memory T cells, subsets that are attributed a key role within a coordinated anti-tumor immune response. In summary, our data constitute pharmacologic SUMOi as a powerful therapy in a subset of BCL causing massive remodeling of the normal B-cell and T-cell compartment.

Evaluation of nanohardness, elastic modulus, and surface roughness of fluoride-releasing tooth colored restorative materials.

Recently, interest in tooth-colored fluoride-releasing dental materials has increased. Although physical and mechanical properties such as surface hardness, elastic modulus and surface roughness of the restorative materials have been investigated, the effect of different immersion media on these properties is still controversial. The aim of this study was to evaluate the nanohardness, elastic modulus and surface roughness of the fluoride release of tooth-colored restorative materials after immersion in acidic beverages. Prepared samples of three restorative materials (a highly viscous glass ionomer (EQUIA Forte; GC, Tokyo, Japan), a compomer (Dyract XP; Dentsply, Weybridge, UK), and a bioactive restorative material (Activa BioACTIVE; Pulpdent, MA, USA)) were randomly divided and immersed in distilled water, a cola and an orange juice for one week. The HYSITRON T1 950 TriboIndenter device (Hysitron, USA) with the Berkovich diamond indenter tip was used for all measurements. The nanohardness and elastic modulus of the samples were measured by applying a force of 6000 µN to five different points on the sample surface. Surface roughness measurements were evaluated on random samples by scanning five random 40 × 40 µm areas. The properties were measured at the initial and one week after immersion. The values of nanohardness, elastic modulus and surface roughness were tested for significant differences using a two-way analysis of variance (ANOVA) with repeated measures (p < 0.05). Tukey's honest significant difference (HSD) test was used for multiple comparisons. AB (Activa BioACTIVE) had the highest initial mean values for nanohardness. After post-immersion, the highest mean value for elastic modulus was the initial AB value. The lowest mean value for roughness of 100.36 nm was obtained for the initial DX (Dyract XP) measurement. Acidic beverages had a negative effect on the nanohardness, elastic modulus and surface roughness of the restorative materials.

Activated SUMOylation restricts MHC class I antigen presentation to confer immune evasion in cancer.

Activated SUMOylation is a hallmark of cancer. Starting from a targeted screening for SUMO-regulated immune evasion mechanisms, we identified an evolutionarily conserved function of activated SUMOylation, which attenuated the immunogenicity of tumor cells. Activated SUMOylation allowed cancer cells to evade CD8+ T cell-mediated immunosurveillance by suppressing the MHC class I (MHC-I) antigen-processing and presentation machinery (APM). Loss of the MHC-I APM is a frequent cause of resistance to cancer immunotherapies, and the pharmacological inhibition of SUMOylation (SUMOi) resulted in reduced activity of the transcriptional repressor scaffold attachment factor B (SAFB) and induction of the MHC-I APM. Consequently, SUMOi enhanced the presentation of antigens and the susceptibility of tumor cells to CD8+ T cell-mediated killing. Importantly, SUMOi also triggered the activation of CD8+ T cells and thereby drove a feed-forward loop amplifying the specific antitumor immune response. In summary, we showed that activated SUMOylation allowed tumor cells to evade antitumor immunosurveillance, and we have expanded the understanding of SUMOi as a rational therapeutic strategy for enhancing the efficacy of cancer immunotherapies.

Duplex DNA engagement and RPA oppositely regulate the DNA-unwinding rate of CMG helicase.

A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.

Dynamics of the Eukaryotic Replicative Helicase at Lagging-Strand Protein Barriers Support the Steric Exclusion Model.

Progression of DNA replication depends on the ability of the replisome complex to overcome nucleoprotein barriers. During eukaryotic replication, the CMG helicase translocates along the leading-strand template and unwinds the DNA double helix. While proteins bound to the leading-strand template efficiently block the helicase, the impact of lagging-strand protein obstacles on helicase translocation and replisome progression remains controversial. Here, we show that CMG and replisome progressions are impaired when proteins crosslinked to the lagging-strand template enhance the stability of duplex DNA. In contrast, proteins that exclusively interact with the lagging-strand template influence neither the translocation of isolated CMG nor replisome progression in Xenopus egg extracts. Our data imply that CMG completely excludes the lagging-strand template from the helicase central channel while unwinding DNA at the replication fork, which clarifies how two CMG helicases could freely cross one another during replication initiation and termination.

The mechanism of DNA unwinding by the eukaryotic replicative helicase.

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.

Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome.

In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.