Quinacrine Has Preferential Anticancer Effects on Mesothelioma Cells With Inactivating NF2 Mutations

Mesothelioma is a rare cancer with disproportionately higher death rates for shipping and mining populations. These patients have few treatment options, which can be partially attributed to limited chemotherapy responses for tumors. We initially hypothesized that quinacrine could be combined with cisplatin or pemetrexed to synergistically eliminate mesothelioma cells. The combination with cisplatin resulted in synergistic cell death and the combination with pemetrexed was not synergistic, although novel artificially-generated pemetrexed-resistant cells were more sensitive to quinacrine. Unexpectedly, we discovered cells with NF2 mutations were very sensitive to quinacrine. This change of quinacrine sensitivity was confirmed by NF2 ectopic expression and knockdown in NF2 mutant and wildtype cell lines, respectively. There are few common mutations in mesothelioma and inactivating NF2 mutations are present in up to 60% of these tumors. We found quinacrine alters the expression of over 3000 genes in NF2-mutated cells that were significantly different than quinacrine-induced changes in NF2 wildtype cells. Changes to NF2/hippo pathway biomarkers were validated at the mRNA and protein levels. Additionally, quinacrine induces a G1 phase cell cycle arrest in NF2-mutated cells versus the S phase arrest in NF2-wildtype cells. This study suggests quinacrine may have repurposing potential for a large subset of mesothelioma patients.

Plasticity in AAA+ proteases reveals ATP-dependent substrate specificity principles

SummaryIn bacteria, AAA+ proteases such as Lon and ClpXP specifically degrade substrates to promote growth and stress responses. Here, we find that an ATP-binding mutant of ClpX suppresses physiological defects of a Lon-deficient strain by shifting ClpXP protease specificity toward normally Lon-restricted substrates and away from normal ClpXP targets. Reconstitution with purified proteins assigns these effects to changes in direct recognition and processing of substrates. We show that wildtype ClpXP specificity can be similarly altered when ATP hydrolysis is reduced, which unexpectedly accelerates degradation of some substrates. This activation corresponds with changes in ClpX conformation, leading to a model where ClpX cycles between ‘capture’ and ‘processive’ states depending on ATP loading. Limiting ATP binding alters dynamics between states affording better recognition of unorthodox substrates, but worse degradation of proteins specifically bound by the processive state. Thus, AAA+ protease specificities can be directly tuned by differences in ATP hydrolysis rates.HighlightsA mutation in the Walker B region of ClpX induces recognition of new substrates.Proteases are optimized for specific functions but barrier to recognize new substrates is easily overcome.Expanding substrate recognition by a protease comes at the cost of reducing native substrate degradation.Decreasing ATP enhances ClpXP mediated degradation of certain classes of substrates.ClpX adopts distinct conformational states to favor better recognition of some substrates over others.Graphical AbstractIn a wildtype cell, AAA+ proteases Lon and ClpXP promote normal growth by degrading distinct substrates. ClpX*P can compensate for the absence of the Lon protease by tuning ClpXP substrate specificity to better degrade Lon-privileged substrates (such as DnaA, SciP, and misfolded proteins) but this comes at the cost of native ClpXP substrates (such as ssrA-tagged proteins and CtrA). We propose that ClpX alternates between a closed and open conformation and promoting one state over the other leads to alterations in substrate specificity. In the presence of ClpX* or in ATP-limited conditions, the open state is favored, allowing capture and recognition of substrates such as casein. The balance shifts to the closed state under high ATP conditions, allowing degradation of substrates such as GFP-ssrA, which preferentially bind the closed state.

Uncoupling of the Diurnal Growth Program by Artificial Genome Relaxation in Synechocystis sp. PCC 6803

In cyanobacteria DNA supercoiling varies over the diurnal light/dark cycle and is integrated with temporal programs of transcription and replication. We manipulated DNA supercoiling in Synechocystis sp. PCC 6803 by CRISPRi-based knock-down of gyrase subunits and overexpression of topoisomerase I, and characterized the phenotypes. Cell division was blocked, most likely due to inhibition of genomic but not plasmid DNA replication. Cell growth continued to 4-5x of the wildtype cell volume, and metabolic flux was redirected towards glycogen in the topoI overexpession strain. Topoisomerase I induction initially lead to down-regulation of GC-rich and up-regulation of AT-rich genes. The response quickly bifurcated and four diurnal co-expression cohorts (dawn, noon, dusk and night) all responded differently, in part with a circadian (≈24 h) pattern. We suggest a model where energy- and gyrase-gated transcription of growth genes at the dark/light transition (dawn) generates DNA supercoiling which then directly facilitates DNA replication and initiates the diurnal transcriptome program.

Expression of the apelinergic system and its influence on functional properties of tumor cells

Small peptide Apelin and its cognate receptor APJ, are known to play a role in tumor angiogenesis and overall cancer progession. Certain authors suggest that the Apelin receptor is also a factor in cancer immunotherapy. In this article, our goal was to study the effects of in vitro targeting of the Apelin/APJ system on the tumor cells functional properties. Protein surface and mRNA expression of Apelin and APJ had been largely examined in various tumor-derived cell lines. In contrast to the tumor tissue, the results of this study demonstrated that most tumor cell lines exhibited somewhat moderate expression of Apelin/APJ. Similar effects of APJ stimulation and inhibition had been observed in in vitro functional assays, which was due to their unusually low expression levels. Low APJ expression in cell lines has been overcome by stable APJ overexpression. In such conditions, stimulation of apelinergic system APJ-overexpressed cells affected cell functional properties in comparison to the wildtype cell lines, where overexpression of APJ receptor resulted in increased migration. On the other hand, no effect on cell proliferation was observed. Consequently, Apelin/APJ signaling in tumor-derived cell lines is not expected to play a direct and crucial role in in vitro cancer survival. Further investigation should focus on in vivo role of the apelinergic system, as demonstrated in the recently published studies, where apelinergic system is claimed to be a promising target for anti-cancer therapy.