The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small-molecule inhibitors and immunotherapy have risen to prominence with chimeric antigen receptor T-cells, checkpoint inhibitors, kinase inhibitors, and monoclonal antibody therapies being deployed across a range of solid organ and haematological malignancies. However, novel approaches are required to target transcription factors and oncogenic fusion proteins that are central to cancer biology and have generally eluded successful drug development. Thalidomide analogues causing protein degradation have been a cornerstone of treatment in multiple myeloma, but a lack of in-depth mechanistic understanding initially limited progress in the field. When the protein cereblon (CRBN) was found to mediate thalidomide analogues\' action and CRBN\'s neo-targets were identified, existing and novel drug development accelerated, with applications outside multiple myeloma, including non-Hodgkin\'s lymphoma, myelodysplastic syndrome, and acute leukaemias. Critically, transcription factors were the first canonical targets described. In addition to broadening the application of protein-degrading drugs, resistance mechanisms are being overcome and targeted protein degradation is widening the scope of druggable proteins against which existing approaches have been ineffective. Examples of targeted protein degraders include molecular glues and proteolysis targeting chimeras (PROTACs): heterobifunctional molecules that bind to proteins of interest and cause proximity-induced ubiquitination and proteasomal degradation via a linked E3 ligase. Twenty years since their inception, PROTACs have begun progressing through clinical trials, with early success in targeting the oestrogen receptor and androgen receptor in breast and prostate cancer respectively. This review explores important developments in targeted protein degradation to both treat and study cancer. It also considers the potential advantages and challenges in the translational aspects of developing new treatments. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Mutations in FBXO7 have been discovered to be associated with an atypical parkinsonism. We report here a new homozygous missense mutation in a paediatric patient that causes an L250P substitution in the dimerisation domain of Fbxo7. This alteration selectively ablates the Fbxo7-PI31 interaction and causes a significant reduction in Fbxo7 and PI31 levels in patient cells. Consistent with their association with proteasomes, patient fibroblasts have reduced proteasome activity and proteasome subunits. We also show PI31 interacts with the MiD49/51 fission adaptor proteins, and unexpectedly, PI31 acts to facilitate SCFFbxo7-mediated ubiquitination of MiD49. The L250P mutation reduces the SCFFbxo7 ligase-mediated ubiquitination of a subset of its known substrates. Although MiD49/51 expression was reduced in patient cells, there was no effect on the mitochondrial network. However, patient cells show reduced levels of mitochondrial function and mitophagy, higher levels of ROS and are less viable under stress. Our study demonstrates that Fbxo7 and PI31 regulate proteasomes and mitochondria and reveals a new function for PI31 in enhancing the SCFFbxo7 E3 ubiquitin ligase activity.

The association of chronic inflammation with colorectal carcinoma (CRC) development is well known in ulcerative colitis (UC). However, the role of inflammatory changes in sporadic CRC pathogenesis is less widely appreciated. In this study, in the first step using RNA-seq, we identified gene-pathway-level changes in UC-associated CRC (UC CRC, n = 10) and used the changes as a proxy for inflammation in human colon to ask if there were associations of inflammatory pathway dysregulations in sporadic CRC pathogenesis (n = 8). We found down-regulations of several inflammation-related metabolic pathways (nitrogen metabolism, sulfur metabolism) and other pathways (bile secretion, fatty acid degradation) in sporadic CRC. Non-inflammation-related changes included up-regulation of the proteasome pathway. In the next step, from a larger number of paired samples from sporadic CRC patients (n = 71) from a geographically and ethnically different population and using a different platform (microarray), we asked if the inflammation-CRC association could be replicated. The associations were significant even after stratification by sex, tumor stage, grade, MSI status, and KRAS mutation status. Our findings have important implications to widen our understanding of inflammatory pathogenesis of sporadic CRC. Furthermore, targeting of several of these dysregulated pathways could provide the basis for improved therapies for CRC.

Glioblastoma, the most severe form of brain tumor and a leading cause of death within a year of diagnosis, is characterized by excessive protein synthesis and folding in the lumen of the endoplasmic reticulum (ER), leading to increased ER stress in the cells of GBM tissues. To mitigate this stress the cancer cells have intelligently adopted a plethora of response mechanisms and Unfolded Protein Response (UPR) is one of those. To bear with this exhaustive situation cells upregulate a strong protein degradation system in form of 26S proteasome and blocking of proteasomal gene synthesis may be a potential therapeutic action against GBM. Proteasomal gene synthesis is exclusively dependent on the transcription factor Nuclear respiratory factor 1 (NRF1) and its activating enzyme DNA damage inducible 1 homolog 2 (DDI2). Here in this study, we performed molecular docking against DDI2 with the 20 FDA-approved drugs and identified Alvimopan and Levocabastine as the top two compounds with the best binding score along with the standard drug Nelfinavir. MD simulation (100 ns) of these protein-ligand docked complexes reveals that the stability and compactness of Alvimopan are high in comparison with Nelfinavir. Our in-silico (Molecular docking and Molecular dynamics simulation) studies pointed out that Alvimopan may be repurposed as a DDI2 inhibitor and can be used as a potential anticancer agent for the treatment of brain tumors.Communicated by Ramaswamy H. Sarma.

The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14 (RPN11), USP14 and UCH37 (UCHL5). However, the functional roles and specificities of these proteasomal DUBs remain elusive. To reveal the specificities of proteasome associated DUBs, we used SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. We observed distinct effects on the global ubiquitinome upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggested less functional redundancy than previously anticipated. We also investigated whether the small molecule inhibitor b-AP15 has the potential to specifically target USP14 and UCH37 by comparing treatment of wild-type versus USP14/UCH37 double-knockout cells with this drug. Strikingly, broad and severe off-target effects were observed, questioning the alleged specificity of this inhibitor. In conclusion, this work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.
Introduction: The removal of (poly)ubiquitin chains at the proteasome is a key step in the protein degradation pathway that determines which proteins are degraded and ultimately decides cell fate. Three different deubiquitinating enzymes (DUBs) are associated to the human proteasome, PSMD14/RPN11, USP14 and UCH37/UCHL5. However, the functional roles and specificities of these proteasomal DUBs remains elusive.
We have applied a SILAC based quantitative ubiquitinomics to study the effects of CRISPR-Cas9 based knockout of each of these DUBs on the dynamic cellular ubiquitinome. Also, we have studied the function of the small molecule inhibitor b-AP15, which has the potential to specifically target USP14 and UCH37.
We report distinct effects on the ubiquitinome and the ability of the proteasome to clear proteins upon removal of either USP14 or UCH37, while the simultaneous removal of both DUBs suggests less redundancy than previously anticipated. In addition, broad and severe off-target effects were observed for b-AP15, questioning the alleged specificity of this inhibitor.
This work presents novel insights into the function of proteasome associated DUBs and illustrates the power of in-depth ubiquitinomics for screening the activity of DUBs and of DUB modulating compounds.

Skin health is heavily affected by ultraviolet irradiation from the sun. In addition, senile skin is characterized by major changes in the collagen, elastin and in the hyaluronan content. Natural products (NPs) have been shown to delay cellular senescence or in vivo aging by regulating age-related signaling pathways. Moreover, NPs are a preferable source of photoprotective agents and have been proven to be useful against the undesirable skin hyperpigmentation. Greek flora harvests great plant diversity with approximately 6000 plant species, as it has a wealth of NPs. Here, we report an extensive screening among hundreds of plant species. More than 440 plant species and subspecies were selected and evaluated. The extracts were screened for their antioxidant and anti-melanogenic properties, while the most promising were further subjected to various in vitro and cell-based assays related to skin aging. In parallel, their chemical profile was analyzed with High-Performance Thin-Layer Chromatography (HPTLC) and/or Ultra-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UPLC-HRMS). A variety of extracts were identified that can be of great value for the cosmetic industry, since they combine antioxidant, photoprotective, anti-melanogenic and anti-aging properties. In particular, the methanolic extracts of Sideritis scardica and Rosa damascena could be worthy of further attention, since they showed interesting chemical profiles and promising properties against specific targets involved in skin aging.

Isatuximab (Isa), an anti-CD38 monoclonal antibody, and carfilzomib (K), a next-generation proteasome inhibitor (PI), both have potent single-agent activity in relapsed and refractory multiple myeloma (RRMM).
This phase 1b study evaluated the combination of Isa and K in 33 patients with RRMM. Isa was administered by intravenous infusion in 3 dosing cohorts: dose level 1 (Isa at 10 mg/kg biweekly), dose level 2 (DL2; Isa at 10 mg/kg weekly for 4 doses and then biweekly), and dose level 3 (Isa at 20 mg/kg weekly for 4 doses and then biweekly) and all patients received K (20 mg/m2 intravenously for cycle 1, days 1 and 2, and then 27 mg/m2 for all subsequent doses). A standard 3+3 dose-escalation design was used, no dose-limiting toxicity was observed, and the maximum tolerated dose was not reached. An expansion cohort of 18 patients was enrolled at DL2 to further evaluate safety and efficacy. Responses were assessed with the International Myeloma Working Group response criteria, and patients continued treatment until disease progression or unacceptable toxicity.
With a median follow-up of 26.7 months, in this heavily pretreated population with a median of 3 prior lines (refractory to PIs and immunomodulatory drugs, 76%; refractory to K, 27%), the overall response rate was 70% (stringent complete response/complete response, 4; very good partial response, 8; partial response, 11). The median progression-free survival was 10.1 months, and the 2-year survival probability was 76%. The most common treatment-related adverse events (grade 2 or higher) were anemia, leukopenia, neutropenia, thrombocytopenia, hypertension, and infection. Infusion reactions were common (55%) but did not limit dosing.
Treatment with Isa plus K was well tolerated with no unexpected toxicity. The combination was effective despite the enrollment of heavily pretreated patients with RRMM.
This phase 1b study was designed to assess the safety, pharmacokinetics, and preliminary efficacy of isatuximab and carfilzomib in patients with relapsed and refractory multiple myeloma. Thirty-three patients were treated: 15 in dose escalation and 18 in dose expansion. Patients received an average of 10 cycles. The treatment was safe and effective. No unexpected toxicity or drug-drug interactions were noted. Seventy percent of the subjects responded to therapy, and the progression-free survival was 10.1 months.

Background and Aims: Part 2 of our ongoing research with anti-angiogenic effects focuses on Wild chrysanthemum; a heat-clearing and detoxicating Traditional Chinese Medicine (TCM). We screened six heat-clearing and detoxicating TCM and noticed that wild chrysanthemum has a potent anti-angiogenic effect in zebrafish. This study aims to determine the genetic mechanisms underlying the anti-angiogenic effects of wild chrysanthemum. Methods: Wild chrysanthemum was decocted, concentrated, sieved and desiccated to attain the water extract. 200μg/mL wild chrysanthemum water extract (WCWE) was diluted in 0.1% dimethyl sulfoxide (DMSO) and given to zebrafish via fish water. 48h post-fertilization (hpf) fli1a-EGFP transgenic zebrafish were used to assay angiogenesis. mRNA-seq, qRT-PCR assay and a parallel reaction monitor (PRM) were carried out to reveal the underlying mechanisms. Results: WCWE showed a significant anti-angiogenic effect in zebrafish. The results of mRNA-seq showed that there were 1119 genes up-regulated and 1332 genes down-regulated by WCWE. The bioinformatic analysis based on mRNA-seq demonstrated that the proteasome signaling pathway was significantly down-regulated. The results of the qRT-PCR assay were consistent with those of the mRNA-seq assay. The results of the PRM assay showed that nine proteins involved in proteasome signaling and the protein expression level of ctnnb2 were significantly down-regulated. The results of the KEGG pathway analysis based on PRM assay demonstrated that WCWE may have an inhibitory action on the regulatory particle of the proteasome. Conclusion: Wild chrysanthemum has a significant anti-angiogenic effect in zebrafish and it may have an inhibitory action on the regulatory particle of the proteasome. The mechanisms underlying the anti-angiogenic effects of wild chrysanthemum may be related to the down-regulation of proteasome/β-catenin signaling in zebrafish.

Strategies for enhancing protein degradation have been proposed for treating neurological diseases associated with a decline in proteasome activity. A proteasomal deubiquitinating enzyme that controls substrate entry into proteasomes, ubiquitin-specific protease 14 (USP14), is an attractive candidate for therapies that modulate proteasome activity. This report tests the validity of genetic and pharmacological tools to study USP14\'s role in regulating protein abundance. Although previous studies implicated USP14 in the degradation of microtubule associate protein tau, tar DNA binding protein, and prion protein, the levels of these proteins were similar in our neurons cultured from wild type and USP14-deficient mice. Neither loss nor over-expression of USP14 affected the levels of these proteins in mice, implying that modifying the amount of USP14 is not sufficient to alter their steady-state levels. However, neuronal over-expression of a catalytic mutant of USP14 showed that manipulating USP14\'s ubiquitin-hydrolase activity altered the levels of specific proteins in vivo. Although pharmacological inhibitors of USP14\'s ubiquitin-hydrolase activity reduced microtubule associate protein tau, tar DNA binding protein, and prion protein in culture, the effect was similar in wild type and USP14-deficient neurons, thus impacting their use for specifically evaluating USP14 in a therapeutic manner. While examining how targeting USP14 may affect other proteins in vivo, this report showed that fatty acid synthase, v-rel reticuloendotheliosis viral oncogene homolog, CTNNB1, and synaptosome associated protein 23 are reduced in USP14-deficient mice; however, loss of USP14 differentially altered the levels of these proteins in the liver and brain. As such, it is critical to more thoroughly examine how inhibiting USP14 alters protein abundance to determine if targeting USP14 will be a beneficial strategy for treating neurodegenerative diseases.

Increasing evidence suggests that prenatal exposure to arsenic, even at common environmental levels, adversely affects child health. These adverse effects include impaired fetal growth, which can carry serious health implications lifelong. However, the mechanisms by which arsenic affects fetal health and development remain unclear.
We addressed this question using a group of 46 pregnant women selected from the New Hampshire Birth Cohort Study (NHBCS), a US cohort exposed to low-to-moderate arsenic levels in drinking water through the use of unregulated private wells. Prenatal arsenic exposure was assessed using maternal urine samples taken at mid-gestation. Samples of the fetal portion of the placenta were taken from the base of the umbilical cord insertion at the time of delivery, stored in RNAlater and frozen. We used RNA sequencing to analyze changes in global gene expression in the fetal placenta associated with in utero arsenic exposure, adjusting for maternal age. Gene set enrichment analysis and enrichment mapping were then used to identify biological processes represented by the differentially expressed genes. Since our previous analyses have identified considerable sex differences in placental gene expression associated with arsenic exposure, we analyzed male and female samples separately.
At FDR < 0.05, no genes were differentially expressed in female placenta, while 606 genes were differentially expressed in males. Genes showing the most significant associations with arsenic exposure in females were LEMD1 and UPK3B (fold changes 2.51 and 2.48), and in males, FIBIN and RANBP3L (fold changes 0.14 and 0.15). In gene set enrichment analyses, at FDR < 0.05, a total of 211 gene sets were enriched with differentially expressed genes in female placenta, and 154 in male placenta. In female but not male placenta, 103 of these gene sets were also associated with reduced birth weight.
Our results reveal multiple biological functions in the fetal placenta that are potentially affected by increased arsenic exposure, a subset of which is sex-dependent. Further, our data suggest that in female infants, the mechanisms underlying the arsenic-induced reduction of birth weight may involve activation of stress response pathways.