PKA is a downstream effector of many inflammatory mediators that induce pain hypersensitivity by increasing the mechanosensitivity of nociceptive sensory afferent. Here, we examine the molecular mechanism underlying PKA-dependent modulation of the mechanically activated ion channel PIEZO2, which confers mechanosensitivity to many nociceptors. Using phosphorylation site prediction algorithms, we identified multiple putative and highly conserved PKA phosphorylation sites located on intracellular intrinsically disordered regions of PIEZO2. Site-directed mutagenesis and patch-clamp recordings showed that substitution of one or multiple putative PKA sites within a single intracellular domain does not alter PKA-induced PIEZO2 sensitization, whereas mutation of a combination of nine putative sites located on four different intracellular regions completely abolishes PKA-dependent PIEZO2 modulation, though it remains unclear whether all or just some of these nine sites are required. By demonstrating that PIEZO1 is not modulated by PKA, our data also reveal a previously unrecognized functional difference between PIEZO1 and PIEZO2. Moreover, by demonstrating that PKA only modulates PIEZO2 currents evoked by focal mechanical indentation of the cell, but not currents evoked by pressure-induced membrane stretch, we provide evidence suggesting that PIEZO2 is a polymodal mechanosensor that engages different protein domains for detecting different types of mechanical stimuli.

Genetic factors are fundamental in the etiology of thoracic aortic aneurysm and dissection (TAAD), but the genetic cause is detected in only about 30% of cases. To define unreported TAAD-associated sequence variants, exome and gene panel sequencing was performed in 323 patients. We identified heterozygous CDKL1 variants [c.427T>C p.(Cys143Arg), c.617C>T p.(Ser206Leu), and c.404C>T p.(Thr135Met)] in 6 patients from 3 families with TAAD spectrum disorders. CDKL1 encodes a protein kinase involved in ciliary biology. Amino acid substitutions were predicted to affect CDKL1 catalytic activity or protein binding properties. CDKL1 was expressed in vascular smooth muscle cells in normal and diseased human aortic wall tissue. Cdkl1 knockdown and transient knockout in zebrafish resulted in intersomitic vessel (ISV) malformations and aortic dilation. Coinjection of human CDKL1 ^wild-type RNA, but not CDKL1 ^Cys143Arg and CDKL1 ^Ser206Leu RNA, rescued ISV malformations. All variants affected CDKL1 kinase function and profiling data, and altered protein-protein binding properties, particularly with ciliary transport molecules. Expression of CDKL1 variants in heterologous cells interfered with cilia formation and length, CDKL1 localization, and p38 MAPK and Vegf signaling. Our data suggest a role of CDKL1 variants in the pathogenesis of TAAD spectrum disorders. The association between primary cilia dysregulation and TAAD expands our knowledge of the underlying molecular pathophysiology.

Radiotherapy (RT) is a central treatment for prostate cancer (PCa), relying on the induction of DNA double-strand breaks (DSBs). Tumor ability to repair these breaks limits RT efficacy, making DSB repair inhibitors potential radiosensitizers. However, many of these inhibitors lack tumor specificity and harm normal cells. Therefore, tumor-specific radiosensitization strategies are critically needed for PCa. Approximately 50% of PCa cases harbor the TMPRSS2-ERG gene fusion, leading to overexpression of the ERG transcription factor (ERG ⁺ ). In this study, we demonstrate that ERG ⁺ tumors shift DSB repair toward the poly(ADP-ribose) polymerase 1–dependent end-joining (PARP1-EJ) pathway. Proteomic and Western blot analyses revealed elevated PARP1, XRCC1, and LIG3 levels in ERG ⁺ cells. Notably, PARP inhibition with olaparib increased residual γH2AX/53BP1 foci postirradiation in ERG ⁺ cells, indicating enhanced radiosensitization. In tissue slice cultures (TSCs) from 53 tumors of patients with high-risk PCa, olaparib selectively increased γH2AX/53BP1 foci selectively in ERG ⁺ samples. ERG ⁺ patient–derived organoids also showed significantly delayed growth when treated with olaparib plus RT, compared with either treatment alone. Interestingly, ERG-negative cells within ERG ⁺ TSCs were similarly radiosensitized by olaparib, likely through bystander effect, with residual 53BP1 foci levels comparable to those in ERG ⁺ cells. This was confirmed by medium exchange experiments. These findings suggest that ERG expression promotes dependency on the PARP1-EJ pathway, rendering ERG ⁺ PCa more susceptible to PARP inhibition. This supports combining PARP inhibitors with RT for tumor-selective radiosensitization in ERG ⁺ patients.

T cell activation critically depends on the calcium ion (Ca ²⁺ )–mobilizing second messenger NAADP (nicotinic acid adenine dinucleotide phosphate), which induces the formation of Ca ²⁺ microdomains that initiate global Ca ²⁺ signals. NAADP is produced in immune synapses in T cells by dual NADPH oxidase 2 (DUOX2). Here, we investigated the mechanisms that stimulate DUOX2 activity in T cells. DUOX2 activity was enhanced by a modest increase in intracellular Ca ²⁺ concentration, similar to that induced by Ca ²⁺ microdomains that arise in resting T cells through different T cell receptor (TCR)–independent mechanisms. In addition, DUOX2 was activated in vitro by phosphorylation of threonine-789 mediated by PKA Cβ or PKCθ, and genetic deficiency of PKA Cβ2 or PKCθ decreased NAADP-dependent Ca ²⁺ microdomain formation in T cells. PKA Cβ2 was activated downstream of adenosine A _2A receptors, independently of the TCR. In contrast, PKCθ was activated by the tyrosine kinase LCK downstream of TCR stimulation. Inhibition of A _2A receptors or PKCθ to prevent full DUOX2 activation decreased the production of the proinflammatory cytokine IL-17 by effector T cells. Thus, full stimulation of NAADP signaling that is critical for T cell activation requires integration of multiple TCR-independent and -dependent signals with different spatiotemporal characteristics by DUOX2, a fine-tuning mechanism that could be relevant for inflammation.

The Warburg effect is a shift from oxidative phosphorylation to anaerobic glycolysis, accompanied by an enormous increase in glucose uptake into cancer cells. We have utilized this effect to design a new group of targeted 1,4-naphthoquinone–glucose derivatives conjugated with a novel thiomethylene linker that are cytotoxic to prostate cancer cells. Compound PeS-9 revealed the highest efficacy and selectivity, which was conditioned by a GLUT-1–mediated uptake. PeS-9 induced androgen receptor degradation followed by downregulation of its signaling. In addition, it increased reactive oxygen species production and induced DNA double-strand breaks. Combinational therapy with PARP inhibitor olaparib resulted in synergistic effects in homologous recombination–deficient cells. The underlying mode of PeS-9’s cytotoxic action involved mitochondrial targeting, leading to a loss of mitochondrial membrane potential, release of cytochrome C and apoptosis-inducing factor, activation of caspases-3 and -9, PARP cleavage, and apoptotic cell death. This process was stipulated by downregulation of several antiapoptotic factors and induction of endoplasmic reticulum stress. Moreover, drug-induced activation of signaling pathway mediated by p38, JNK1/2, and ERK1/2 kinases was identified as an important factor of the cytotoxic activity. The anticancer activity of PeS-9 could be confirmed ex vivo using patient-derived tumoroids as well as in vivo in xenografts, demonstrating suppression of tumor growth and decreased dissemination of prostate cancer cells to the lungs. No serious side effects were observed in animal models. This unique combination of anticancer properties makes PeS-9 an attractive candidate for targeted monotherapy against GLUT-1–overexpressing tumors and as a potential combination partner, especially with PARP inhibitors.

Background: Remarkable differences exist in the outcome of systemic cancer therapies. Lymphomas and leukemias generally respond well to systemic chemotherapies, while solid cancers often fail. We engineered different human cancer cells lines to uniformly express a modified herpes simplex virus thymidine kinase TK.007 as a suicide gene when ganciclovir (GCV) is applied, thus in theory achieving a similar response in all cell lines. Methods: Fifteen different cell lines were engineered to express the TK.007 gene. XTT-cell proliferation assays were performed and the IC₅₀-values were calculated. Functional kinome profiling, mRNA sequencing, and bottom-up proteomics analysis with Ingenuity pathway analysis were performed. Results: GCV potency varied among cell lines, with lymphoma and leukemia cells showing higher susceptibility than solid cancer cells. Functional kinome profiling implies a contribution of the SRC family kinases and decreased overall kinase activity. mRNA sequencing highlighted alterations in the MAPK pathways and bottom-up proteomics showed differences in apoptotic and epithelial junction signaling proteins. Conclusions: The histogenetic origin of cells influenced the susceptibility of human malignant cells towards cytotoxic agents with leukemias and lymphomas being more sensitive than solid cancer cells.