Microtubules (MTs) are dynamic cytoskeletal structures essential for intracellular transport, cell division, and organelle positioning. Their functions are regulated by post-translational modifications, including alpha-tubulin acetylation at Lys40, which enhances MT stability and resilience. Histone deacetylase 6 (HDAC6) is the primary enzyme that reverses this modification, but its access to the luminal Lys40 residue is restricted. Previously, we identified SETDB1, a histone methyltransferase and known oncogene, as a cytoplasmic regulator of MT dynamics, attenuating MT polymerization and destabilizing MTs. Here, we uncover the molecular mechanism by which SETDB1 destabilizes MTs. SETDB1 interacts with HDAC6 and promotes its tubulin deacetylation activity. Mechanistically, SETDB1 enhances HDAC6 recruitment to polymerized MTs and induces repairable damage along MT shafts, generating entry points for HDAC6 into the MT lumen. Functionally, this axis regulates Golgi organization: SETDB1 overexpression disperses the Golgi in an HDAC6-dependent manner, while SETDB1 knockdown or HDAC6 inhibition compacts it. Notably, SETDB1 role in Golgi regulation is independent of its methyltransferase activity. These findings reveal crosstalk among the histone methylation machinery, MT dynamics, and Golgi organization. Since Golgi dispersal is thought to promote tumorigenesis, our results suggest that the SETDB1-HDAC6 axis is a potential therapeutic target.

Translesion synthesis (TLS) is a DNA damage tolerance pathway that enables cells to replicate across damaged DNA, thereby promoting cell survival under genotoxic stress, however, contributing to genomic instability and therapy resistance. C#3 is a small molecule that disrupts the interaction between MAD2L2 and Rev1, a key complex required for DNA polymerase ζ-mediated TLS. Here, we further investigated the impact of C#3 on MAD2L2-dependent TLS activity and cellular responses to DNA damage. Consistent with our previous findings, C#3 enhanced cellular sensitivity to cisplatin in multiple cancer cell lines and increased DNA damage signaling following treatment. Using a non-replicating plasmid assay, we demonstrate that C#3 impairs lesion bypass across DNA adducts in mammalian cells. Disruption of the MAD2L2-Rev1 axis was further supported by reduced formation of MAD2L2-Rev1 complexes, as assessed by proximity ligation assay. Functionally, cells exposed to C#3 during recovery from cisplatin treatment exhibited persistent γH2AX signaling, consistent with delayed resolution of replication-associated DNA damage. In-vivo, combined treatment with C#3 and cisplatin reduced tumor growth in syngeneic melanoma and triple-negative breast cancer mouse models compared with either treatment alone. Together, these findings demonstrate that pharmacological disruption of the MAD2L2-Rev1 axis impairs TLS associated DNA damage tolerance, enhances cellular responses to cisplatin-induced DNA damage, and suppresses tumor growth in-vivo. These results support targeting MAD2L2-dependent TLS as a potential strategy for improving the efficacy of DNA damaging chemotherapy.

Thyroid hormones (THs) regulate metabolism, proliferation, and genomic stability. Clinical studies have linked levothyroxine therapy with higher Oncotype DX Recurrence Scores in breast cancer (BC), suggesting a potential effect of thyroid hormone signaling on genomic risk. Here, we investigated the impact of triiodothyronine (T3) on DNA damage and repair pathways in estrogen receptor-positive T47D breast cancer and non-tumorigenic MCF10A cells. RNA sequencing revealed significant upregulation of RAD51 and enrichment of DNA repair pathways following 24 h T3 exposure. Consistently, T3 increased γH2AX and 53BP1 nuclear foci, indicating transient activation of the DNA damage response (DDR). These effects were transient, returning to baseline after 48 h, suggesting cellular adaptation. T3 also enhanced proliferation at 10 μM but inhibited growth at higher concentrations. Our findings indicate that acute exposure to T3 induces transient genomic stress, providing a potential mechanistic basis for the observed association between thyroid hormone therapy and increased BC recurrence risk.

DNA-damaging platinum-based chemotherapeutics (cisplatin, carboplatin, oxaliplatin) have been the first line of treatment for cancer for decades. They bind or modify DNA and block replication, generating cytotoxicity and apoptosis of rapidly dividing cancer cells. Translesion synthesis (TLS), a DNA damage tolerance pathway under normal conditions, enables cancerous cells to tolerate DNA damage by employing mutagenic replication at DNA lesion sites. Consequently, chemotherapy promotes the formation of treatment-based mutations, which can lead to chemotherapy-resistant secondary malignancies, increasing morbidity in many cancer patients.

MAD2L2 is a small HORMA domain protein that plays a crucial role in DNA repair and mitosis. In both TLS and shieldin, the dimerization of MAD2L2 via its HORMA domain is critical for the stability and function of these complexes. However, in mitosis, the dimerization state of MAD2L2 remains unknown. To assess the importance of MAD2L2’s dimerization during mitosis, we utilized CRISPR/Cas9 to generate MAD2L2 knockout cells, which were subsequently complemented with MAD2L2 species carrying different dimer-disrupting point mutations. We assessed the ability of these MAD2L2 dimer-disrupting mutants to regulate mitosis by evaluating early mitotic events and mitotic fidelity. Our findings indicate that MAD2L2 can function in its monomeric form during mitosis, suggesting that MAD2L2 homodimerization is dispensable for early mitotic regulation. Furthermore, our results suggest that the binding of CDH1 to MAD2L2 is a key regulating factor in mitosis that may actively prevent the formation of MAD2L2 dimers, thereby shifting the cellular balance toward MAD2L2-CDH1 interaction. Thus, the equilibrium between the monomeric and dimeric forms of MAD2L2 is an important cellular factor regulating the MAD2L2-containing complexes.

OBJECTIVES:SETDB1 is a methyltransferase responsible for the methylation of histone H3-lysine-9, which is mainly related to heterochromatin formation. SETDB1 is overexpressed in various cancer types and is associated with an aggressive phenotype. In agreement with its activity, it mainly exhibits a nuclear localization; however, in several cell types a cytoplasmic localization was reported. Here we looked for cytoplasmic functions of SETDB1.METHODS:SETDB1 association with microtubules was detected by immunofluorescence and co-sedimentation. Microtubule dynamics were analysed during recovery from nocodazole treatment and by tracking microtubule plus-ends in live cells. Live cell imaging was used to study mitotic kinetics and protein-protein interaction was identified by co-immunoprecipitation.RESULTS:SETDB1 co-sedimented with microtubules and partially colocalized with microtubules. SETDB1 partial silencing led to faster polymerization and reduced rate of catastrophe events of microtubules in parallel to reduced proliferation rate and slower mitotic kinetics. Interestingly, over-expression of either wild-type or catalytic dead SETDB1 altered microtubule polymerization rate to the same extent, suggesting that SETDB1 may affect microtubule dynamics by a methylation-independent mechanism. Moreover, SETDB1 co-immunoprecipitated with HDAC6 and tubulin acetylation levels were increased upon silencing of SETDB1.CONCLUSIONS:Taken together, our study suggests a model in which SETDB1 affects microtubule dynamics by interacting with both microtubules and HDAC6 to enhance tubulin deacetylation. Overall, our results suggest a novel cytoplasmic role for SETDB1 in the regulation of microtubule dynamics.

DNA-damaging chemotherapy agents such as cisplatin have been the first line of treatment for cancer for decades. While chemotherapy can be very effective, its long-term success is often reduced by intrinsic and acquired drug resistance, accompanied by chemotherapy-resistant secondary malignancies. Although the mechanisms causing drug resistance are quite distinct, they are directly connected to mutagenic translesion synthesis (TLS). The TLS pathway promotes DNA damage tolerance by supporting both replication opposite to a lesion and inaccurate single-strand gap filling. Interestingly, inhibiting TLS reduces both cisplatin resistance and secondary tumor formation. Therefore, TLS targeting is a promising strategy for improving chemotherapy. MAD2L2 (i.e., Rev7) is a central protein in TLS. It is an essential component of the TLS polymerase zeta (ζ), and it forms a regulatory complex with Rev1 polymerase. Here we present the discovery of two small molecules, c#2 and c#3, that directly bind both in vitro and in vivo to MAD2L2 and influence its activity. Both molecules sensitize lung cancer cell lines to cisplatin, disrupt the formation of the MAD2L2-Rev1 complex and increase DNA damage, hence underlining their potential as lead compounds for developing novel TLS inhibitors for improving chemotherapy treatments.

Purpose: We aimed to examine how various types of cancer, classified histologically, affect semen quality. Methods: The study group included 313 patients who were diagnosed with cancer and reached for a sperm cryopreservation before a gonadotoxic treatment (PG-Tx group). Their semen parameters were compared to those of two control groups: (a) individuals who attended a fertility investigation and were found to be above the limit of the lower reference value of the WHO 2010 manual (ARL group), and (b) fertile men, whose semen parameters were obtained from the dataset of the WHO 2020 manual. Results: Semen quality was significantly poorer in the PG-Tx group than in the ARL group. Differences included a 65.6% decrease in concentration, a 12.1% decrease in volume, a 72.7% decrease in total count, and a 33.0%, 22.2%, and 24.7% decrease in total motility, rapid motility, and progressive motility, respectively. Linear regression models comparing the PG-Tx and ARL groups revealed that the maximum reduction in total motility and concentration was in men with germ-cell tumors, whereas the minimum reduction was in hematological tumors. Similarly, all sperm quality parameters were significantly lower in the PG-Tx group than in the fertile-men group (p < 0.0001). Conclusions: While the effect of malignancy on semen parameters is debatable, we found that all examined types of cancer significantly impaired sperm quality parameters. Although the median of most semen parameters of patients with cancer were still in the normal WHO range, their fifth percentile, represents men with a delayed time to pregnancy.

BACKGROUND/AIM:Germline mutations in PTCH1 or SUFU in the sonic hedgehog (SHH) pathway cause Gorlin's syndrome with increased risk of developing SHH-subgroup medulloblastoma. Gorlin's syndrome precludes the use of radiotherapy (a standard component of treatment) due to the development of multiple basal cell carcinomas. Also, current SHH inhibitors are ineffective against SUFU-mutated medulloblastoma, as they inhibit upstream genes. In this study, we aimed to detect differences in the expression of genes and microRNAs between SUFU- and PTCH1-mutated SHH medulloblastomas which may hint at new treatment directions.PATIENTS AND METHODS:We sequenced RNA and microRNA from tumors of two patients with germline Gorlin's syndrome - one having PTCH1 mutation and one with SUFU mutation - followed by bioinformatics analysis to detect changes in genes and miRNAs expression in these two tumors. Expression changes were validated using qRT-PCR. Ingenuity pathway analysis was performed in search for targetable pathways.RESULTS:Compared to the PTCH1 tumor, the SUFU tumor demonstrated lower expression of miR-301a-3p and miR-181c-5p, matrix metallopeptidase 11 (MMP11) and OTX2, higher expression of miR-7-5p and corresponding lower expression of its targeted gene, connexin 30 (GJB6). We propose mechanisms to explain the phenotypic differences between the two types of tumors, and understand why PTCH1 and SUFU tumors tend to relapse locally (rather than metastatically as in other medulloblastoma subgroups).CONCLUSION:Our results help towards finding new treatable molecular targets for these types of medulloblastomas.

MAD2L2 (i.e. Rev7) is a central regulatory protein important in several processes, such as translesion synthesis (TLS), DNA damage response and mitosis. In TLS, MAD2L2 binds Rev3 to form Pol zeta (ζ) and promotes formation of the Pol ζ- REV1 complex allowing extension beyond distorted DNA structures. MAD2L2 is also part of the heterotetrameric shieldin complex that regulates DNA repair at sites of damage, where similarly to TLS, it bridges the interaction between SHLD2 and SHLD3. Lastly, during mitosis, MAD2L2 prevents premature activation of the anaphase promoting complex/cyclosome (APC/C), by sequestering its activator, CDH1. MAD2L2 exits in a 'closed' active conformation binding Rev3 and Rev1, or SHLD2 and SHLD3, and an 'open' inactive conformation, with no binding partners. Moreover, Pol ζ- REV1 forms a homodimer using a protein-protein interaction (PPI) domain comprised of a central αC helix, promoting Rev3-MAD2L2 interaction and C-terminus β-sheets, enabling Rev1-MAD2L2 interaction. While the role of MAD2L2 in TLS is well established, molecular details regarding the CDH1-MAD2L2 interaction and MAD2L2 homodimerization are still missing. Here we demonstrate, in a human cell line, using a series of MAD2L2 mutants, that MAD2L2's C-terminus interface is essential for the CDH1-MAD2L2 binding as well as for homodimerization. In addition, we show that CDH1 interacts with MAD2L2 in a Rev1-like pattern, using the same C-terminus residues on MAD2L2 which Rev1 binds. Thus, identification of CDH1 as an additional Rev1-like binding protein strengthens the versatility of MAD2L2 as a regulatory protein and emphasizes the complexity involved in MAD2L2's preferential complex formation.

Background/Aim: Accurate regulation of the spindle assembly checkpoint (SAC) and anaphase promoting complex/cyclosome (APC/C) are essential for the correct execution of mitosis. In this work, we focused on MAD2L2 (REV7), a central translesion (TLS) protein, which also functions as a mitotic regulator by inhibiting APC/C in prometaphase. Materials and Methods: Using bioinformatics analysis, live cell imaging and APC/C protein binding and degradation assays, we explored the influence of MAD2L2 over-expression in breast cancer. Results: A significant over-expression of MAD2L2 was found in triple negative breast cancers (TNBC), compared to other breast cancers, correlating to poor patient prognosis. We also identified significant over-expression of MAD2L2 in the MDA-MB-157 triple negative (TN) cell line. A high percentage of MDA-MB-157 cells failed to complete mitosis and died during mitosis or shortly after. In addition, these cells completed mitosis at a significantly slower rate than control cells. MDA-MB-157 cells present high levels of mitotic slippage upon nocodazole treatment and acute dysregulation in APC/C function and substrate degradation. Moreover, silencing of MAD2L2 in the MDA-MB-157 cell line improved mitotic phenotypes. Conclusion: MAD2L2 over-expression supports the carcinogenic phenotype of MDA-MB-157 cells by promoting uncontrolled mitosis.

REV7, also termed mitotic arrest?deficient 2-like 2 (MAD2L2 or MAD2B), acts as an interaction module in a broad array of cellular pathways, including translesion DNA synthesis, cell cycle control, and nonhomologous end joining. Numerous REV7 binding partners have been identified, including the human small GTPase Ras-associated nuclear protein (RAN), which acts as a potential upstream regulator of REV7. Notably, the Shigella invasin IpaB hijacks REV7 to disrupt cell cycle control to prevent intestinal epithelial cell renewal and facilitate bacterial colonization. However, the structural details of the REV7?RAN and REV7?IpaB interactions are mostly unknown. Here, using fusion protein and rigid maltose-binding protein tagging strategies, we determined the crystal structures of these two complexes at 2.00?2.35 ? resolutions. The structures revealed that both RAN and IpaB fragments bind the ?safety belt? region of REV7, inducing rearrangement of the C-terminal ?-sheet region of REV7, conserved among REV7-related complexes. Of note, the REV7-binding motifs of RAN and IpaB each displayed some unique interactions with REV7 despite sharing consensus residues. Structural alignments revealed that REV7 has an adaptor region within the safety belt region that can rearrange secondary structures to fit a variety of different ligands. Our structural and biochemical results further indicated that REV7 preferentially binds GTP-bound RAN, implying that a GTP/GDP-bound transition of RAN may serve as the molecular switch that controls REV7's activity. These results provide insights into the regulatory mechanism of REV7 in cell cycle control, which may help with the development of small-molecule inhibitors that target REV7 activity.

Unlike histone H3, which is present only in S phase, the variant histone H3.3 is expressed throughout the cell cycle [1] and is incorporated into chromatin independent of replication [2]. Recently, H3.3 has been implicated in the cellular response to ultraviolet (UV) light [3]. Here, we show that chicken DT40 cells completely lacking H3.3 are hypersensitive to UV light, a defect that epistasis analysis suggests may result from less-effective nucleotide excision repair. Unexpectedly, H3.3-deficient cells also exhibit a substantial defect in maintaining replication fork progression on UV-damaged DNA, which is independent of nucleotide excision repair, demonstrating a clear requirement for H3.3 during S phase. Both the UV hypersensitivity and replication fork slowing are reversed by expression of H3.3 and require the specific residues in the α2 helix that are responsible for H3.3 binding its dedicated chaperones. However, expression of an H3.3 mutant in which serine 31 is replaced with alanine, the equivalent residue in H3.2, restores normal fork progression but not UV resistance, suggesting that H3.3[S31A] may be incorporated at UV-damaged forks but is unable to help cells tolerate UV lesions. Similar behavior was observed with expression of H3.3 carrying mutations at K27 and G34, which have been reported in pediatric brain cancers. We speculate that incorporation of H3.3 during replication may mark sites of lesion bypass and, possibly through an as-yet-unidentified function of the N-terminal tail, facilitate subsequent processing of the damage.

The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/C-CDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/C-CDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/C-CDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/C-CDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/C-CDC20 and APC/C-CDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity.

Our knowledge concerning the mechanisms of cell cycle regulation in organisms belonging to the Trypanosometidae family is limited. Leishmania donovani are parasitic protozoa that cause kala azar, a fatal form of visceral leishmaniasis in humans. Here we provide evidence that the L. donovani genome contains a Cdc20 homologue. Cdc20 is a regulator of the Anaphase Promoting Complex/Cyclosome (APC/C) that mediates ubiquitin-dependent proteasomal degradation of key cell cycle regulators in eukaryotes. We show that L. donovani Cdc20 protein (LdCdc20p) can complement a lack of yeast Cdc20 protein in Saccharomyces cerevisiae cells, validating the functionality of LdCdc20p. Furthermore, we demonstrate cyclic expression of LdCdc20p and that it contains an active RXXL destruction motif, a distinctive feature of proteins targeted for proteasomal degradation by APC/C. Finally, in line with the proteasome mediating LdCdc20p degradation, promastigotes exposed to proteasome inhibitor display elevated LdCdc20p levels. Taken together our data indicate that Leishmania regulate their cell cycle by ubiquitin-dependent proteasomal degradation mediated by the APC/C.

The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time.

The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1–cyclin B is thus an inhibitor, activator, and substrate of the cyclosome.

The cyclosome pathway of ubiquitin-mediated proteolysis plays an essential role in cell cycle control. The multisubunit cyclosome is regulated by transient interactions with Fizzy (Fzy) and Fizzy-related (Fzr) genes. We report here that both Fzy and Fzr are transcribed in a cell cycle specific but distinct manner. Fzy transcription starts after the restriction point in late G1 and ceases upon cell division. Fzr transcription also ceases upon cell division but resumes already in mid G1, before the restriction point, and takes place also in G0. Fzr has further a striking cell cycle specific pattern of mRNA stability. During most of the cell cycle its message is fairly stable, however upon exit from mitosis it is rapidly degraded. This result is puzzling because Fzr is essential for cyclosome activity in G1, and points to a complex pattern of Fzr regulation.

Exit from mitosis in eukaryotic cells is regulated by the cyclosome (also called anaphase promoting complex or APC), a multisubunit ubiquitin ligase that acts on mitotic cyclins. Previous studies in a cell-free system from clam oocytes have shown that the activation of the cyclosome at the end of mitosis involves its phosphorylation by protein kinase Cdk1/cyclin B. Genetic and biochemical studies have furthermore indicated that cyclosome activity also requires a WD-40 repeat containing protein called Fizzy (FZY) or Cdc20. It has been suggested [Fang et al. (1998) Mol. Cell 2, 163-171] that in the presence of FZY, the phosphorylation of the cyclosome is not critical for its activation. By contrast, we find that the activity of the interphase, non-phosphorylated form of the cyclosome from clam embryos is not stimulated by FZY to a significant extent. However, when interphase cyclosome is first incubated with protein kinase Cdk1/cyclin B, the subsequent supplementation of FZY greatly stimulates its cyclin-ubiquitin ligase activity. Furthermore, phosphatase treatment of purified mitotic cyclosome prevents its stimulation by FZY, a process that can be reversed by the action of protein kinase Cdk1/cyclin B. We conclude that in the early embryonic cell cycles, the primary event in the activation of the cyclosome at the end of mitosis is its Cdk1-dependent phosphorylation and activation by FZY takes place in a subsequent process.