Renata Tisi

BackgroundKRAS mutations are major oncogenic drivers, yet state-independent pan-KRAS inhibitors capable of targeting both active (GTP-bound) and inactive (GDP-bound) states remain extremely scarce.PurposeThe purpose of this work is to evaluate the non-covalent, state-independent pan-KRAS inhibitory potential of rosmarinic acid (RA) and botanical extracts from Rosmarinus officinalis and Salvia officinalis.Study designOur integrated multidisciplinary approach combines chemical characterization (qNMR and UPLC-ESI-HRMS/MS), biophysical binding assays (STD-NMR), computational analyses (molecular docking and molecular dynamics simulations), and in vitro functional assays on KRAS-dependent cancer cell lines, and Caco-2 cell monolayers as a non-proliferating epithelial model.MethodsMetabolites were profiled by NMR and UPLC-ESI-HRMS/MS and RA was quantified using qNMR. Direct interaction of RA and extracts with KRAS in GDP- and GMPPCP-bound states was characterized by STD-NMR, while molecular docking was used to investigate the predicted binding sites of RA. Antiproliferative activity and selectivity were assessed on KRAS-mutant cancer cells versus quiescent Caco-2 monolayers.ResultsqNMR provided accurate quantification of RA in both extracts. STD-NMR demonstrated direct, non-covalent, and state-independent binding of RA to KRAS in both inactive and active-like states, while docking analyses supported the proposed binding modes. Purified RA and botanical extracts inhibited the proliferation of KRAS-mutant cancer cells while showing limited cytotoxicity toward non-proliferating Caco-2 epithelial monolayers.ConclusionRosmarinic acid represents a promising non-covalent, state-independent pan-KRAS lead candidate, supporting further investigation of its potential for KRAS-targeted drug development.

In eukaryotes, DNA double-strand breaks (DSBs) activate Tel1/ATM- and Mec1/ATR-dependent checkpoint signaling and can be repaired by homologous recombination (HR), which requires DNA end resection, typically initiated by the MRX/MRN complex. In budding yeast, Sae2/CtIP promotes MRX-dependent end processing, limits Tel1–Rad53 signaling, and contributes to DSB end-tethering. Here, we identify a Tel1 FAT-domain variant, Tel1-400, that increases the DNA damage resistance of sae2Δ cells by suppressing their DSB end-tethering defect through Rad9 phosphorylation-dependent oligomerization. Tel1-400 accumulates more strongly at DSBs than wild-type Tel1 and enhances DNA-damage-induced Rad9 phosphorylation. An oligomerization-defective rad9 phosphosite mutant prevents Tel1-400 from improving DSB end-tethering in sae2Δ cells, whereas chemically induced Rad9 dimerization restores this ability. Rad9 also supports DSB end-tethering in Sae2-proficient cells, as the same rad9 phosphosite mutant increases end separation in an otherwise wild-type background. Functionally, Tel1-400 shifts repair in sae2Δ cells toward strand-invasion-dependent HR while reducing single-strand annealing, and this repair-pathway bias requires Rad9 oligomerization. Together, these findings identify a Tel1–Rad9 pathway in which increased Tel1 association at DNA breaks enhances Rad9 phosphorylation and self-assembly, thereby promoting DSB end-tethering and influencing recombination pathway choice.

Yeast Sae2 plays a dual role in the DNA damage response by suppressing Rad53 activation and stimulating DNA end clipping via the MRX complex. Using AlphaFold3-based modeling and mutational analysis, here we show that Mec1/Tel1-dependent phosphorylation of Sae2 at T90 or T279 is sufficient to restrain Rad9-Rad53 interaction and Rad53 kinase activation. Cells expressing a non-phosphorylatable Sae2 double mutant (T90A T279A) display persistent Rad53 activation, whereas phosphomimetic Sae2 variants (T90E or T279E) restore normal checkpoint inactivation. Structural modeling and charge-reversal genetics indicate that electrostatic interactions between phosphorylated T90/T279 of Sae2 and Rad53 residue R70 are critical for this regulation. In addition, T279 phosphorylation, but not T90, cooperates with cyclin-dependent kinase (CDK)-dependent phosphorylation of Sae2 S267 to promote MRX-dependent resolution of hairpin DNA structures and processing of meiotic double-strand breaks (DSBs). A Sae2 T279E phosphomimetic partially rescues both hairpin cleavage defects and DNA damage sensitivity of tel1Δ cells, indicating that Tel1 promotes MRX activity primarily through Sae2 T279 phosphorylation.

The yeast Sae2 protein, known as CtIP in mammals, once phosphorylated at Ser267, stimulates the endonuclease activity of the Mre11-Rad50-Xrs2 (MRX) complex to cleave DNA ends that possess hairpin structures or protein blocks, such as the Spo11 transesterase or trapped topoisomerases. Stimulation of the Mre11 endonuclease by Sae2 depends on a Rad50–Sae2 interaction, but the mechanism by which this is achieved remains to be elucidated. Through genetic studies, we show that the absence of the last 23 amino acids from the Sae2 C-terminus specifically impairs MRX-dependent DNA cleavage events, while preserving the other Sae2 functions. Employing AlphaFold3 protein structure predictions, we found that the Rad50–Sae2 interface involves not only phosphorylated Ser267 but also the phosphorylated Thr279 residue and the C-terminus of Sae2. This region engages in multiple interactions with residues that are mutated in rad50-s mutants, which are known to be specifically defective in the processing of Spo11-bound DNA ends. These interactions are critical for stabilizing the association between Sae2 and Rad50, thereby ensuring the correct positioning of Mre11 in its active endonucleolytic state.

The yeast Rif2 protein is known to inhibit Mre11 nuclease and the activation of Tel1 kinase through a short motif termed MIN, which binds the Rad50 subunit and simulates its ATPase activity in vitro. The mechanism by which Rif2 restrains Tel1 activation and the consequences of this inhibition at DNA double-strand breaks (DSBs) are poorly understood. In this study, we employed AlphaFold-Multimer modelling to pinpoint and validate the interaction surface between Rif2 MIN and Rad50. We also engineered the rif2-S6E mutation that enhances the inhibitory effect of Rif2 by increasing Rif2-Rad50 interaction. Unlike rif2Δ, the rif2-S6E mutation impairs hairpin cleavage. Furthermore, it diminishes Tel1 activation by inhibiting Tel1 binding to DSBs while leaving MRX association unchanged, indicating that Rif2 can directly inhibit Tel1 recruitment to DSBs. Additionally, Rif2^S6E reduces Tel1-MRX interaction and increases stimulation of ATPase by Rad50, indicating that Rif2 binding to Rad50 induces an ADP-bound MRX conformation that is not suitable for Tel1 binding. The decreased Tel1 recruitment to DSBs in rif2-S6E cells impairs DSB end-tethering and this bridging defect is suppressed by expressing a Tel1 mutant variant that increases Tel1 persistence at DSBs, suggesting a direct role for Tel1 in the bridging of DSB ends.

DNA double-strand breaks (DSBs) are a significant threat to cell viability due to the induction of genome instability and the potential loss of genetic information. One of the key players for early DNA damage response is the conserved Mre11/Rad50 Nbs1/Xrs2 (MRN/X) complex, which is quickly recruited to the DNA’s ruptured ends and is required for their tethering and their subsequent repair via different pathways. The MRN/X complex associates with several other proteins to exert its functions, but it also exploits sophisticated internal dynamic properties to orchestrate the several steps required to address the damage. In this review, we summarize the intrinsic molecular features of the MRN/X complex through biophysical, structural, and computational analyses in order to describe the conformational transitions that allow for this complex to accomplish its multiple functions.

Telomere biology disorders (TBDs) are characterized by short telomeres, premature aging, bone marrow failure and cancer predisposition. Germline mutations in NHP2, encoding for one component of the telomerase cofactor H/ACA RNA binding complex together with Dyskerin, NOP10 and GAR1, have been previously reported in rare cases of TBDs. Here, we report two novel NHP2 variants (NHP2-A39T and NHP2-T44M) identified in a compound heterozygous patient affected by premature aging, bone marrow failure/myelodysplastic syndrome and gastric cancer. Although still able to support cell viability, both variants reduce the levels of hTR, the telomerase RNA component, and telomerase activity, expanding the panel of NHP2 pathological variants. Furthermore, both variants fail to be incorporated in the H/ACA RNA binding complex when in competition with wild-type endogenous NHP2, and the lack of incorporation causes their drastic proteasomal degradation. By RoseTTAFold prediction followed by molecular dynamics simulations, we reveal a dramatic distortion of residues 33–41, which normally position on top of the NHP2 core, as the main defect of NHP2-A39T, and high flexibility and the misplacement of the N-terminal region (residues 1–24) in NHP2-T44M and, to a lower degree, in NHP2-A39T. Because deletion of amino acids 2–24 causes a reduction in NHP2 levels only in the presence of wild-type NHP2, while deletion of amino acids 2–38 completely disrupts NHP2 stability, we propose that the two variants are mis-incorporated into the H/ACA binding complex due to the altered dynamics of the first 23 amino acids and/or the distortion of the residues 25–41 loop.

DNA double-strand breaks (DSBs) can be repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ). NHEJ is induced by the binding to DSBs of the Ku70–Ku80 heterodimer, which acts as a hub for the recruitment of downstream NHEJ components. An important issue in DSB repair is the maintenance of the DSB ends in close proximity, a function that in yeast involves the MRX complex and Sae2. Here, we provide evidence that Ku contributes to keep the DNA ends tethered to each other. The ku70-C85Y mutation, which increases Ku affinity for DNA and its persistence very close to the DSB ends, enhances DSB end-tethering and suppresses the end-tethering defect of sae2Δ cells. Impairing histone removal around DSBs either by eliminating Tel1 kinase activity or nucleosome remodelers enhances Ku persistence at DSBs and DSB bridging, suggesting that Tel1 antagonizes the Ku function in supporting end-tethering by promoting nucleosome removal and possibly Ku sliding inwards. As Ku provides a block to DSB resection, this Tel1 function can be important to regulate the mode by which DSBs are repaired.

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has been discussed for its safety and efficacy in cancer treatments. For this reason, we have inquired into its use on triple-negative human breast cancer. Analyzing the biological effects of CBD on MDA-MB-231, we have demonstrated that both CBD dosage and serum concentrations in the culture medium influence its outcomes; furthermore, light scattering studies demonstrated that serum impacts the CBD aggregation state by acting as a surfactant agent. Pharmacological studies on CBD in combination with chemotherapeutic agents reveal that CBD possesses a protective action against the cytotoxic effect exerted by cisplatin on MDA-MB-231 grown in standard conditions. Furthermore, in a low serum condition (0.5%), starting from a threshold concentration (5 µM), CBD forms aggregates, exerts cytostatic antiproliferative outcomes, and promotes cell cycle arrest activating autophagy. At doses above the threshold, CBD exerts a highly cytotoxic effect inducing bubbling cell death. Finally, IGF-1 and EGF antagonize the antiproliferative effect of CBD protecting cells from harmful consequences of CBD aggregates. In conclusion, CBD effect is strongly associated with the physical state and concentration that reaches the treated cells, parameters not taken into account in most of the research papers.