Inflammatory bowel disease is characterized by unresolved mucosal inflammation driven mainly by TNFα and/or interferon gamma (IFNγ). While anti-TNFα biologics are a clinical mainstay, therapeutic resistance remains a significant hurdle. The molecular mechanisms that sustain inflammation in anti-TNFα nonresponders are not fully understood. In human colon biopsies, we identified significant upregulation of the deubiquitinase OTUD5 in nonresponders compared to responders, suggesting its involvement in therapy resistance. Using an intestinal epithelial cell -specific Otud5 KO mouse model, we show that Otud5 deficiency significantly alleviated the IFNγ-dependent colitis induced by dextran sulfate sodium, as evidenced by reduced weight loss and diminished infiltration of Ly6C+ inflammatory monocytes. Mechanistically, IFNγ induces OTUD5 expression through a nontranscriptional mechanism; in turn, OTUD5 stabilizes STAT1 and STAT2 by preventing their ubiquitination and subsequent degradation. This sustains IFNγ-ISGF3 signaling, which directly drives the expression of CCL8, a critical chemokine for monocyte recruitment. Targeting this pathway with a newly identified small-molecule inhibitor, CT1170, which exhibits potent activity against OTUD5, blocked the IFNγ-ISGF3-CCL8 axis, halted colitis progression, and suppressed colitis-elicited tumorigenesis. These findings were validated in human inflammatory bowel disease organoids, where CT1170 effectively disrupted IFNγ-driven inflammatory signaling.

Esophageal squamous cell carcinoma (ESCC) remains a lethal malignancy with limited therapeutic options. The deubiquitinase USP28 has emerged as a key stabilizer of the oncogenic transcription factor ΔNp63 in squamous cancers, yet its functional significance and therapeutic potential in ESCC are unexplored. Here, we elucidate that USP28 is essential for ESCC proliferation. Genetic ablation of USP28 induced profound G2/M cell cycle arrest and apoptosis, phenotypes mechanistically linked to the destabilization of ΔNp63. We further establish that USP28 directly binds to and deubiquitinates ΔNp63, thereby controlling its protein stability. Crucially, targeting this axis with CT1113, a novel and potent USP28 inhibitor, recapitulated the anti-tumor effects of genetic knockdown, triggering ΔNp63 degradation, cell cycle arrest, and apoptosis in ESCC cells. Importantly, CT1113 administration significantly suppressed tumor growth in ESCC xenograft models. Our study not only defines the USP28/ΔNp63 axis as a critical driver of ESCC but also validates the therapeutic strategy of pharmacologically inhibiting USP28 for the treatment of this aggressive cancer.

The sense of taste is essential as it governs appetite and the feeding process. However, it is largely unknown whether and how tumors in a host communicate with peripheral taste sensing. Using the well-established ykiS168A tumor models in Drosophila melanogaster, we found that flies carrying brain or gut tumors exhibit enhanced avoidance of bitter compounds such as caffeine but showed no avoidance of sucrose, and the degree of the avoidance was correlated with severity of the tumor phenotype. Through RNAi screening of upregulated cytokines secreted by malignant tumors, we identified Upd3 as a key factor in this process. Tumor-derived Upd3 promotes systematic increase of Spz5 expression, a fly neurotrophin, which in turn activates the Toll-6 receptor in peripheral bitter sensing neurons, leading to upregulation of the bitter-sensing receptor Gr66a. In vivo Ca2+ imaging demonstrated heightened responses to caffeine by Gr66a+ neurons in tumor-bearing flies. Interestingly, a similar phenomenon was observed in murine tumor models, where tumors also caused behavioral hypersensitivity to bitter tastants. Our findings reveal how tumors affect animals' feeding behavior as well as the underlying mechanism. Our findings also suggest that such tumor-induced behavioral alterations are likely conserved across species.

Introduction Anti-CD20 monoclonal antibodies are widely used in the treatment of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL). These therapies often lead to good initial responses, but many patients eventually relapse. One known cause of resistance is the decreased or lost expression of CD20 on tumor cells. Notably, suppression of the oncogenic transcription factor c-Myc has been associated with increased CD20 expression [1]. USP28 (ubiquitin-specific protease 28) is a deubiquitinating enzyme that stabilizes multiple oncogenic substrates, including c-Myc, by preventing their proteasomal degradation. Our team has developed CT1113, a selective USP28 inhibitor, which has been evaluated in a Phase I clinical trial for patients with acute myeloid leukemia [2]. This study aims to determine whether USP28 inhibition can suppress c-Myc, upregulate CD20 expression, and enhance the efficacy of anti-CD20 immunotherapy. Materials and Methods Human DLBCL and BL cell lines (OCI-Ly1, OCI-Ly10, Raji, and Daudi) were treated with CT1113 or subjected to USP28 knockdown via shRNA. CD20 mRNA and protein expression levels were assessed using quantitative RT-PCR, flow cytometry, and western blotting. For in vivo studies, a xenograft model was established by intravenous injection of luciferase-expressing Daudi cells into NCG (NOD/ShiLtJGptPrkdcem26Cd52Il2rgem26Cd22/Gpt) mice. Tumor burden was monitored using the IVIS Imaging System. Mice received vehicle, CT1113, Rituximab, or combination treatment. Results CT1113 treatment and USP28 knockdown both resulted in significant downregulation of c-Myc protein and upregulation of CD20 mRNA and protein expression in BL and DLBCL cell lines. Proteomic analyses further confirmed a robust increase in CD20 expression following CT1113 treatment. In vivo, combination therapy with CT1113 and Rituximab significantly suppressed tumor burden in the spleen and bone marrow, demonstrating superior antitumor activity compared to monotherapy. These data suggest that USP28 inhibition upregulates CD20 through c-Myc suppression and enhances anti-CD20 therapeutic efficacy. Conclusion These findings demonstrate that targeting of USP28 leads to c-Myc suppression and CD20 upregulation, thereby enhancing the response to anti-CD20 immunotherapy in c-Myc–driven B-cell lymphomas. The observed synergistic antitumor activity supports further clinical investigation of CT1113 in combination with Rituximab in patients with relapsed or refractory disease. References Torun, Anna et al. “Potassium/sodium cation carriers robustly upregulate CD20 antigen by targeting MYC, and synergize with anti-CD20 immunotherapies to eliminate malignant B cells.” Haematologica vol. 110,7 (2025): 1555-1572. Peng, Jin et al. “Identification of a class of potent USP25/28 inhibitors with broad-spectrum anti-cancer activity.” Signal transduction and targeted therapy vol. 7,1 393. 8 Dec. 2022.

The RAD51 recombinase is evolutionarily conserved critical for homologous recombination (HR)-mediated repair of DNA double-strand breaks. It binds to single strand DNA to form protein-DNA filaments for homology searching and pairing during HR repair. RFWD3 is an E3 ubiquitin ligase shown to remove RAD51 at the completion of HR repair through ubiquitination and degradation of RAD51. However, it remains elusive what prevents RFWD3 from attacking RAD51 in the absence of DNA damage and early on during the repair process. Here, we show that it is UHRF1 that protects RAD51, and it does so by acting as an E3 ubiquitin ligase of RFWD3 is demonstrated. Interestingly, RAD51 also protects RFWD3 from UHRF1, thereby establishing a negative feedback circuit that regulates the protein levels of RFWD3 and RAD51. Furthermore, it is shown that the ubiquitination of RFWD3 is regulated by phosphorylation status of UHRF1, and that phosphatase PP4 is important for modulating UHRF1 activity. Altogether, these regulatory mechanisms ensure that the recombinase RAD51 is maintained at appropriate levels for HR repair.

Reductive carboxylation is critical for the proliferation of cancer cells and the differentiation of T cells. However, the role of this reaction in cancer cell-mediated tumor immunity remains unclear. Analysis of TCGA database showed a negative correlation between IDH2 expression and the presence of CD8+ T cells in lung and breast cancers, whereas IDH1 expression didn't show such a correlation. Further GSEA analysis revealed a significant enrichment of immune-related genes within IDH2-associated genes, specifically those in the type Ⅰ interferon pathway. In lung cancer cells, the depletion of IDH2 expression indeed could induce the activation of the immune-related and specially type Ⅰ interferon pathway. Targeting IDH2 with shRNA or its inhibitor AGI-6780 caused an increase in intracellular α-ketoglutarate concentration and a decrease in ATP and SAM levels, leading to a reduction in the methylation of STING promoter and elevated levels expression of STING. The increase of STING expression underlies the activation of type I interferon pathway observed in IDH2 compromised tumor cells and increased defense responses in the tumors in mice. These results identify IDH2 as a potential target to enhance cancer immune therapy.

The deubiquitinase USP28 plays an important role in maintaining the expression levels of a large number of onco-proteins prominent among which is c-MYC. Previous work has shown the potential of USP28 as a pan-cancer therapeutic target. We identified a potent and specific inhibitor of the deubiquitinase, CT1113, which showed excellent preclinical anti-tumor activities. The inhibitor can induce c-MYC degradation in cancer cell lines with all different tissue-origins and in xenograft tumors. We therefore launched a first-in-human phase I trial of CT1113 in patients with relapsed/refractory acute myeloid leukemia (R/R AML). The compound showed excellent safety and PK profiles. Importantly, preliminary efficacy was observed, validating USP28 as a therapeutic target. A phase II trial has been planned. Jie Jin, Xingnong Ye, Huafeng Wang, Xiang Shen, Jin Peng, Pumin Zhang. The first-in-human trial of USP28 inhibitor CT1113 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr CT142.

KRAS is a prominent oncogene mutated in a large number of human malignancies, particularly in pancreatic, colorectal, and lung tumors. We demonstrate here that KRAS, including its various activating mutants, is subjected to ubiquitin-mediated proteasomal degradation in cancer cells. Through an siRNA-based screening of deubiquitinases, we identified USP25 as a deubiquitinase for KRAS. Depleting USP25 expression increases ubiquitination and proteasomal degradation of KRAS, leading to the suppression of its oncogenic activity. We further show that USP25 inhibitors we have discovered are capable of destabilizing KRAS in cancer cells and are efficacious in blocking tumor xenograft growth in mice. These findings provide evidence supporting the notion that targeting the deubiquitinase USP25 can effectively, albeit indirectly, suppress KRAS and potentially aid in the treatment of tumors driven by KRAS-activating mutations.

Metabolic dysfunction-associated steatohepatitis (MASH), a progressive form of metabolic dysfunction-associated fatty liver disease (MAFLD), is a leading cause of liver disease worldwide and can progress to cirrhosis and cancer. Despite its prevalence, the pathogenesis of MASH remains poorly understood, and there is only one U.S. Food and Drug Administration-approved treatment, highlighting the need for new therapeutic strategies. Peroxisome proliferator-activated receptor (PPAR)γ is activated in the liver under high-fat or obese conditions, promoting lipid storage and contributing to MASH progression. We found that USP28 expression is elevated in the livers of MAFLD/MASH patients. Through dietary induction, including a methionine-choline deficient (MCD) diet and a western diet (WD) combined with carbon tetrachloride (CCl4) injections, we established two severe mouse models of MASH to explore the role of USP28. Mechanistically, the hepatic deubiquitinase (DUB) USP28 directly binds to PPARγ, preventing its ubiquitination and subsequent degradation, thereby maintaining the integrity of the PPARγ signaling pathway. In the absence of Usp28 or if the DUB is inhibited, PPARγ is downregulated, and the PPAR signaling pathway is inhibited, enhancing cellular defenses against excess fat. Both genetic and pharmacological inactivation of Usp28 significantly reduced MASH severity induced by the MCD diet or WD-CCl4 regimen, as well as WD-CCl4-induced hepatocellular carcinoma in mice.

Immune checkpoint inhibitor (ICI) has reshaped the landscape of cancer therapy and improved the prognosis of a number of malignancies. However, a considerable proportion of cancer patients fail to benefit from ICI treatment. The main reason for the failure is the poor infiltration of lymphocytes due to the immunosuppressive milieu that dictates a “cold” tumor microenvironment. The ubiquitin-proteasome system plays crucial roles in many cellular processes including tumor progression and immune evasion. USP25 and USP28, two homologous deubiquitinases, maintain the stability of a wide range of oncoproteins by suppressing their proteasomal degradation. We previously found that CT1113, a novel dual USP25/28 inhibitor, could induce a potent and broad-spectrum anti-tumor effect. Here, we demonstrate that CT1113 triggers a robust cytotoxic CD8+ T cell-dependent anti-tumor immune response in several types of “cold” tumors. Dual inhibition of tumoral USP25/28 reverses the immunosuppressive microenvironment by increasing CD8+ T cell infiltration/activity and attenuates the priming of protumoral M2-like macrophages through flow cytometry and single-cell RNA-seq (scRNA-seq). Mechanistically, we identified YTH domain-containing family protein 2 (YTHDF2) as a substrate of USP25/28. The two deubiquitinases redundantly promote the stability of YTHDF2. Dual inhibition of the two increases the degradation of YTHDF2 and abolishes YTHDF2-mediated complement activation and immunosuppression. As a result, both pharmacologic and genetic inhibition of USP25/28 strongly enhance anti-PD-1 therapy against two murine “cold” tumor models without discernable systemic toxicity. Together, these results provide a rationale for future clinical trials evaluating the synergy between inhibiting USP25/28 and ICI to improve the efficacy of immunotherapy. Shujie Zhou, Jiarui Liu, Limin Chen, Jin Peng, Xinghua Zhen, Lingzhi Wu, Xupeng Bai, Pumin Zhang. CT1113, a dual USP25/28 inhibitor, promotes antitumor immunity by preventing YTHDF2-mediated complement activation and potentiates anti-PD-1 therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB367.

Animals have evolved the ability to detect ambient temperatures, allowing them to search for optimal living environments. In search of the molecules responsible for cold-sensing, we examined a Gal4 insertion line in the larvae of Drosophila melanogaster from previous screening work, which has a specific expression pattern in the cooling cells (CCs). We identified that the targeted gene, fa2h, which encodes a fatty acid 2-hydroxylase, plays an important role in cool temperature sensing. We found that fa2h mutants exhibit defects in cool avoidance behavior and that this phenotype could be rescued by genetically re-introducing the wild-type version of FA2H in CCs but not the enzymatically disabled point mutation version. Calcium imaging data showed that CCs require fa2h to respond to cool temperature. Lipidomic analysis revealed that the 2-hydroxy sphingolipids content in the cell membranes diminished in fa2h mutants, resulting in increased fluidity of CC neuron membranes. Furthermore, in mammalian systems, we showed that FA2H strongly regulates the function of the TRPV4 channel in response to its agonist treatment and warming. Taken together, our study has uncovered a novel role of FA2H in temperature sensing and has provided new insights into the link between membrane lipid composition and the function of temperature-sensing ion channels.

T-cell acute lymphoblastic leukaemia (T-ALL) is a highly aggressive and heterogeneous lymphoid malignancy with poor prognosis in adult patients. Aberrant activation of the NOTCH1 signalling pathway is involved in the pathogenesis of over 60% of T-ALL cases. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase known to regulate the stability of NOTCH1. Here, we report that genetic depletion of USP28 or using CT1113, a potent small molecule targeting USP28, can strongly destabilize NOTCH1 and inhibit the growth of T-ALL cells. Moreover, we show that USP28 also regulates the stability of sterol regulatory element binding protein 1 (SREBP1), which has been reported to mediate increased lipogenesis in tumour cells. As the most critical transcription factor involved in regulating lipogenesis, SREBP1 plays an important role in the metabolism of T-ALL. Therefore, USP28 may be a potential therapeutic target, and CT1113 may be a promising novel drug for T-ALL with or without mutant NOTCH1.

Burkitt lymphoma (BL) is a highly aggressive non-Hodgkin lymphoma characterized by IGH/MYC gene translocation, high expression of c-Myc and large lipid droplets in the cytoplasm. The therapeutic options for adult patients with BL remain limited, with intensive multiagent chemotherapy serving as the primary treatment modality. Despite this, the cure rate remains suboptimal. Consequently, the identification and development of novel targeted therapies are imperative to enhance the prognosis of this historically challenging disease. Ubiquitin-specific protease 28 (USP28) is a deubiquitinase that has been reported to facilitate the evasion of ubiquitination and subsequent proteasomal degradation for lots of oncogenic substrates, with c-Myc being one of the identified substrates. Furthermore, our previous studies have shown that USP28 can modulate the SREBP1-mediated lipid synthesis pathway. Our team has developed a small molecule inhibitor, CT1113, which effectively targets USP28 and has been under evaluation in a phase I clinical trial involving patients with acute myeloid leukemia (AML) since 2022. Consequently, it is imperative to investigate the cytotoxic effects of CT1113 on BL cells through experiments and elucidate its underlying mechanisms. This research aims to provide novel therapeutic avenues for the clinical treatment of BL. We first found that compared with diffuse large B-cell lymphoma (DLBCL), BL had up-regulated levels of c-Myc and SREBP1 proteins, and activated lipid synthesis pathways. To investigate the role of USP28 in BL, we conducted loss-of-function experiments in BL cell lines (Raji and Daudi). We observed that the knockdown of USP28 resulted in a substantial reduction in endogenous c-Myc and SREBP1 protein levels, significantly slowed cell proliferation, and induced apoptosis in both Raji and Daudi cells. Subsequently, we investigated the effects of pharmacologic inhibition of USP28 using the inhibitor CT1113 in Raji and Daudi cells. Treatment with CT1113 was found to decrease cell viability, induce apoptosis, and markedly reduce the protein levels of USP28, c-Myc, and SREBP1. Furthermore, BODIPY staining experiments revealed that CT1113 treatment significantly diminished the neutral lipid droplets within the cell plasma. To assess the in vivo efficacy of CT1113, a human xenograft model was established using Raji cells. Luciferase-expressing Raji cells were intravenously injected into NCG mice, which were randomized into two groups: one receiving vehicle treatment and the other receiving CT1113 (20 mg/kg body weight, administered twice daily via oral gavage). CT1113 demonstrated significant suppression of tumorigenesis in the spleen and bone marrow. Notably, CT1113 exhibited dose-dependent cytotoxicity in primary BL cells isolated from the bone marrow of two BL patients. To investigate the potential role of SREBP1 in BL, we administered the SREBP1 inhibitor fatostatin to Raji and Daudi cell lines. The findings indicated that fatostatin reduced cell viability and induced apoptosis in a dose-dependent manner. Furthermore, bioimaging data obtained following the xenograft procedure demonstrated that fatostatin significantly impeded tumor progression in a CDX model established with luciferase-expressing Raji cells. These experimental results suggest that SREBP1-mediated lipid synthesis plays a crucial role in BL, and that pharmacological inhibition of SREBP1 exerts cytotoxic effects on BL cell lines. In summary, our findings demonstrate that CT1113 exhibits significant efficacy against BL. Our preclinical studies of CT1113 indicate that targeting USP28 represents a highly promising and potential clinical strategy for the treatment of BL patients.

Generating a transgene with a reporter inserted into the genome helps us study endogenous gene expression patterns in model organisms. Here, using Drosophila melanogaster, , we present a protocol for generating a P2A-Gal4 insertion through CRISPR-Cas9-mediated homology recombination. We describe the design strategy, steps for constructing the injection plasmids, and the fly-cross scheme for screening the transformants from the G0 generation. This protocol can also be applied to introduce mutations or various genetic tools into the fly genome. For complete details on the use and execution of this protocol, please refer to Li et al.

Host cell-encoded deaminases act as antiviral restriction factors to impair viral replication and production through introducing mutations in the viral genome. We sought to understand whether deaminases are involved in SARS-CoV-2 mutation and replication, and how the viral factors interact with deaminases to trigger these processes. Here, we show that APOBEC and ADAR deaminases act as the driving forces for SARS-CoV-2 mutagenesis, thereby blocking viral infection and production. Mechanistically, SARS-CoV-2 nucleocapsid (N) protein, which is responsible for packaging viral genomic RNA, interacts with host deaminases and co-localizes with them at stress granules to facilitate viral RNA mutagenesis. N proteins from several coronaviruses interact with host deaminases at RNA granules in a manner dependent on its F17 residue, suggesting a conserved role in modulation of viral mutagenesis in other coronaviruses. Furthermore, mutant N protein bearing a F17A substitution cannot localize to deaminase-containing RNA granules and leads to reduced mutagenesis of viral RNA, providing support for its function in enhancing deaminase-dependent viral RNA editing. Our study thus provides further insight into virus-host cell interactions mediating SARS-CoV-2 evolution.

Breast cancer has emerged as the most common cancer globally, with a significant reduction in overall survival rate after metastasis. Compared with other types of breast cancer, triple-negative breast cancer (TNBC) is more prone to metastasize, presenting substantial treatment challenges due to the lack of effective therapies. LGR4, which is highly expressed in breast cancer, has been shown to promote the proliferation and invasion of breast cancer cells. However, its specific role in TNBC remains unclear. In this study, we applied a multi-omics approach to explore the regulatory mechanism of LGR4 in TNBC metastasis. Our findings showed that LGR4 could regulate actin cytoskeletal through EGFR and curtail EGFR activation-induced TNBC metastasis by inhibiting MGP expression. These insights provide new perspectives on the role of LGR4 in breast cancer metastasis.

RAD51 is related to the bacterial RecA protein and is best known for its role in homologous recombination-mediated repair of DNA damage. Here, we report an unexpected function of RAD51 in the maintenance methylation of genomic DNA, a function that is separable from its role in homologous recombination. First, it acts as an inhibitor of the E3 ubiquitin ligase UHRF1. Deficiency in RAD51 causes excessive ubiquitination and degradation of the DNA methyltransferase DNMT1, leading to the loss of global DNA methylation. Second, RAD51 helps UHRF1 to monoubiquitinate histone H3 to generate DNMT1 recruiting signal. It binds H3 directly, enabling UHRF1 to bind and ubiquitinate H3 more readily. Disrupting the interaction between RAD51 and H3 diminishes DNMT1 recruitment and the failure of maintenance methylation of genomic DNA. Thus, RAD51 dually regulates UHRF1. These results establish RAD51 as a guardian of the integrity of both the genome and the epigenome.

Background Triple-negative breast cancer (TNBC) has pronounced stemness that is associated with relapse. N-6-methyladenosine (m(6)A) plays a crucial role in shaping cellular behavior by modulating transcript expression. However, the role of m(6)A in TNBC stemness, as well as the mechanisms governing its abundance, has yet to be elucidated. Methods We analyzed proteomic and transcriptomic data derived from breast cancer cohorts, with an emphasis on m(6)A regulators. To unravel the role of m(6)A in TNBC, we employed RNA sequencing, methylated RNA immunoprecipitation sequencing, RNA immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assays with mesenchymal stem-like (MSL) TNBC models. The clinical relevance was validated using human tissue microarrays and publicly accessible databases. Results Our findings indicate that the global level of m(6)A modification in MSL TNBC is downregulated primarily due to the loss of methyltransferase-like 14 (METTL14). The diminished m(6)A modification is crucial for the maintenance of TNBC stemness, as it increases the expression of yes-associated protein 1 (YAP1) by blocking YTH domain-containing family protein 2 (YTHDF2)-mediated transcript decay, thereby promoting the activation of Hippo-independent YAP1 signaling. YAP1 is essential for sustaining the stemness regulated by METTL14. Furthermore, we demonstrated that the loss of METTL14 expression results from lysine-specific demethylase 1 (LSD1)-mediated removal of histone H3 lysine 4 methylation at the promoter region, which is critical for LSD1-driven stemness in TNBC. Conclusion These findings present an epi-transcriptional mechanism that maintains Hippo-independent YAP1 signaling and plays a role in preserving the undifferentiated state of TNBC, which indicates the potential for targeting the LSD1-METTL14 axis to address TNBC stemness.