SUMOylation Inhibitors Research Articles

The SUMO Family: Mechanisms and Implications in Thyroid Cancer Pathogenesis and Therapy.

(1) Background: Small ubiquitin-like modifiers (SUMOs) are pivotal in post-translational modifications, influencing various cellular processes, such as protein localization, stability, and genome integrity. (2) Methods: This review explores the SUMO family, including its isoforms and catalytic cycle, highlighting their significance in regulating key biological functions in thyroid cancer. We discuss the multifaceted roles of SUMOylation in DNA repair mechanisms, protein stability, and the modulation of receptor activities, particularly in the context of thyroid cancer. (3) Results: The aberrant SUMOylation machinery contributes to tumorigenesis through altered gene expression and immune evasion mechanisms. Furthermore, we examine the therapeutic potential of targeting SUMOylation pathways in thyroid cancer treatment, emphasizing the need for further research to develop effective SUMOylation inhibitors. (4) Conclusions: By understanding the intricate roles of SUMOylation in cancer biology, we can pave the way for innovative therapeutic strategies to improve outcomes for patients with advanced tumors.

Intra-tumoral YAP and TAZ heterogeneity drives collective NSCLC invasion that is targeted by SUMOylation inhibitor TAK-981

Intra-tumoral YAP and TAZ heterogeneity drives collective NSCLC invasion that is targeted by SUMOylation inhibitor TAK-981

Abstract B043: Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration

Abstract Background: Identification of effective immunomodulatory strategies remains a major unmet need in pancreatic ductal adenocarcinoma (PDAC). Agonistic anti-CD40 antibody (aCD40) exhibits direct cytotoxicity on tumor cells and augments antigen presentation. Subasumstat(SUB), a sumoylation inhibitor, augments innate and adaptive immune responses by increasing interferon signaling. We hypothesized that SUB+aCD40 combination would promote an effective antitumor immune response in an aggressive orthotopic model of PDAC. Methods: 2000 KPC 46 cells were implanted in the pancreas of four groups of F1 hybrid (initial survival, CD8+ T-cell depletion, and clodronate liposome depletion) and nude mice, enrolled when tumors had reached 3-5mm. The vehicle (veh) group received 80ul SUB vehicle (q48 hrs) and 100 ug blank IgG (qwk); SUB only group received 15mg/kg q48h; aCD40 only group received 100ug qwk; SUB+aCD40 group received 15mg/kg SUB q48h and 100 ug aCD40 qwk. Single cell RNA sequencing (scRNASeq) was conducted on n=2 tumors from each group. Immunohistochemistry (IHC) was performed on veh and SUB+aCD40 tumors for macrophage infiltration using F4/80. Flow cytometry (FC) was employed for the presence of lymphoid and myeloid markers in veh, SUB+aCD40, and clodronate depleted SUB+aCD40 (SUB+aCD40+clod) groups. Results: Mice receiving SUB+aCD40 had a significantly improved median survival over those receiving monotherapy or vehicle (24.5d vs. SUB:15.5d, aCD40:20d, veh: 19d). scRNASeq revealed increased tumor infiltrating CD8+ T-cell and myeloid populations when comparing veh and monotherapy to SUB+aCD40. F4/80 IHC confirmed increased macrophage infiltration in SUB+aCD40 vs veh (p = 0.01). The survival advantage conferred by SUB+aCD40 was preserved in both CD8+ T-cell depleted mice (p=0.025) and nude mice (p=0.037), while clodronate eliminated the treatment survival advantage (p=0.39). FC showed increased tumor macrophage/monocyte infiltration in SUB+aCD40 vs veh (veh:11.9%, SUB+aCD40:22.5%, p=0.03) and reduction in SUB+aCD40+clod (12.6%, p=0.01). CD8+ T-cell CD69 expression decreased in the SUB+aCD40+clod group (SUB+aCD40:19%, SUB+aCD40+clod:6%, p< 0.01). CD8 T-cell infiltration (SUB+aCD40:4.95%, SUB+aCD40+clod:3.85%, p=0.27) and CD4/CD8 T-cell ratio (veh: 1.26, SUB+aCD40:0.52, SUB+aCD40+clod:0.69, p=0.09) were not affected by clodronate. Conclusion: SUB+aCD40 therapy confers a survival advantage in the KPC46 model of PDAC in a T-cell independent manner by increasing macrophage/monocyte infiltration. SUB+aCD40 promotes CD8+ T-cell infiltration and activation, but they do not contribute to survival advantage, possibly due to exhaustion. Future work will include addition of therapeutics to mitigate/reverse T-cell exhaustion to harness both innate and adaptive immunity in treating PDAC. Citation Format: Asimina Courelli, Kevin Li, James Lee, Herve Tiriac, Yuan Chen, Andrew Lowy. Synergy of Subasumstat and anti-CD40 improves survival by augmenting tumor macrophage infiltration [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B043.

Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization

DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.

RREB1-mediated SUMOylation enhancement promotes chemoresistance partially by transcriptionally upregulating UBC9 in colorectal cancer.

Chemoresistance is a main cause of chemotherapy failure and tumor recurrence. The effects of global protein SUMOylation on chemoresistance in colorectal cancer (CRC) remains to be investigated. Herein, we have proposed that the elevated SUMO2/3-modified proteins confer 5-fluorouracil (5-FU) chemoresistance acquisition in CRC. The SUMOylation levels of global proteins in CRC cell lines were elevated compared with normal colon cell line NCM460. 5-FU treatment obviously reduced SUMOylation of global proteins in 5-FU-sensitive CRC cells including HT29, HCT116 and HCT-8. However, in 5-FU-resistant HCT-8/5-FU cells, the expression level of SUMO2/3-modified proteins was increased under 5-FU exposure in a concentration-dependent manner. 5-FU treatment combined with SUMOylation inhibitor ML-792 significantly increased the sensitivity of 5-FU-resistant cells to 5-FU and reduced colony formation numbers in HCT-8/5-FU cells. And UBC9-mediated SUMOylation elevation contributes to 5-FU resistance in HCT116 cells. Moreover, we also identified RREB1 as a regulator of SUMOylation profiling of global cellular proteins via directly binding to the promoter of UBC9. Overexpression of RREB1 promoted 5-FU resistance in CRC, which was partially abolished by treatment of inhibitor ML-792. In conclusion, RREB1-enhanced protein SUMOylation contributes to 5-FU resistance acquisition in CRC.

SUMO promotes DNA repair protein collaboration to support alternative telomere lengthening in the absence of PML.

The alternative lengthening of telomeres (ALT) pathway maintains telomere length in a significant fraction of cancers that are associated with poor clinical outcomes. A better understanding of ALT mechanisms is therefore necessary for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins contributes to the formation of ALT telomere-associated PML bodies (APBs), in which telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, it is still unknown whether-and if so, how-SUMO supports ALT beyond APB formation. Here, we show that SUMO condensates that contain DNA repair proteins enable telomere maintenance in the absence of APBs. In PML knockout ALT cell lines that lack APBs, we found that SUMOylation is required for manifesting ALT features independent of PML and APBs. Chemically induced telomere targeting of SUMO produces condensate formation and ALT features in PML-null cells. This effect requires both SUMOylation and interactions between SUMO and SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are associated with the accumulation of DNA repair proteins, including Rad52, Rad51AP1, RPA, and BLM, at telomeres. Furthermore, Rad52 can undergo phase separation, enrich SUMO at telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that SUMO condensate formation promotes collaboration among DNA repair factors to support ALT telomere maintenance without PML. Given the promising effects of SUMOylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

Renal protection after hemorrhagic shock in rats: Possible involvement of SUMOylation

Hemorrhagic shock (HS), a leading cause of preventable death, is characterized by severe blood loss and inadequate tissue perfusion. Reoxygenation of ischemic tissues exacerbates organ damage through ischemia–reperfusion injury. SUMOylation has been shown to protect neurons after stroke and is upregulated in response to cellular stress. However, the role of SUMOylation in organ protection after HS is unknown. This study aimed to investigate SUMOylation-mediated organ protection following HS. Male Wistar rats were subjected to HS (blood pressure of 40 ± 2 mmHg, for 90 min) followed by reperfusion. Blood, kidney, and liver samples were collected at various time points after reperfusion to assess organ damage and investigate the profile of SUMO1 and SUMO2/3 conjugation. In addition, human kidney cells (HK-2), treated with the SUMOylation inhibitor TAK-981 or overexpressing SUMO proteins, were subjected to oxygen and glucose deprivation to investigate the role of SUMOylation in hypoxia/reoxygenation injury. The animals presented progressive multiorgan dysfunction, except for the renal system, which showed improvement over time. Compared to the liver, the kidneys displayed distinct patterns in terms of oxidative stress, apoptosis activation, and tissue damage. The global level of SUMO2/3 in renal tissue was also distinct, suggesting a differential role. Pharmacological inhibition of SUMOylation reduced cell viability after hypoxia-reoxygenation damage, while overexpression of SUMO1 or SUMO2 protected the cells. These findings suggest that SUMOylation might play a critical role in cellular protection during ischemia–reperfusion injury in the kidneys, a role not observed in the liver. This difference potentially explains the renal resilience observed in HS animals when compared to other systems.

UBE2I regulates the nuclear translocation of hnRNPA2B1 by contributing to SUMO modification in osteoarthritis

BackgroundOsteoarthritis (OA) is a progressive condition affecting the joints that lacking effective therapy. However, the underlying molecular mechanism has not been fully clarified. MethodsA model of OA was established in Sprague-Dawley (SD) rats through intra-articularly injected with monoiodoacetate (MIA). Western blot analysis was used to identify the levels of UBE2I and hnRNPA2B1 in articular cartilage. Overexpression and siRNA vectors for UBE2I were constructed and transfected into rat chondrocytes. CCK-8, TUNEL and transwell assay were utilized to assess the cell viability, apoptosis and migration ability. Western blot analysis was used to determine the levels of chondrogenic-specific genes including SOX9, COL2A1, Aggrecan, and PRG4. Then, molecular interactions were confirmed by immunoprecipitation. ResultsWe observed significant upregulation of UBE2I and hnRNPA2B1 expression in articular cartilage samples of OA. The Pearson correlation analysis revealed positive correlation between UBE2I and hnRNPA2B1 levels. Functional experiments showed that increased UBE2I expression significantly suppressed cell growth, migration, and reduced the expression of chondrogenic-specific genes, while decreasing UBE2I levels had the opposite effects. Molecular interactions between UBE2I and hnRNPA2B1were determined via co-localization and immunoprecipitation. SUMO1 and SUMO3 proteins were enriched by immunoprecipitation using hnRNPA2B1 antibodies. Rescue experiments were performed using SUMOylation inhibitor (2-D08) and SUMOylation activator (N106). Overexpression of UBE2I increased the expression of hnRNPA2B1 in the cytoplasm and decreased the level in the nucleus, which was reversed by the treatment of 2-D08. Conversely, UBE2I knockdown and N106 treatment had the opposite effect. ConclusionsUBE2I modulated the nuclear translocation of hnRNPA2B1 by promoting SUMOylation in OA.

DIPG-16. THE BIG AND SMALL OF PROTEIN POST-TRANSLATIONAL MODIFICATIONS: TARGETING SUMOYLATION AND CITRULLINATION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Protein post-translational modifications (PTMs) alter the properties of protein through proteolytic cleavage, or the addition of modifying groups to one or more amino acids. Histones are major targets of PTMs, including through SUMOylation of lysine residues and citrullination of arginine residues, with both processes able to regulate gene transcription. We aimed to delineate the potential role of SUMOylation and citrullination in the epigenetic dysregulation that drives Diffuse Midline Glioma (DMG) tumorigenicity. METHODS AND RESULTS DMG tumours demonstrated an upregulation of SUMOylation machinery pathway components and increased expression of peptidylarginine deiminases (PADs), the enzymes responsible for citrullination. Nanomolar doses of the SUMOylation inhibitor TAK-981 blocks DMG cell cycle G2/M transition and results in potent decreases in cell viability and clonogenic potential, triggering ‘viral mimicry’ in DMG cells, resulting in increased expression of interferon stimulated genes and inflammatory mediators including IL-1β, IL-6 and TNFα, whilst also causing a pro-inflammatory M1 macrophage response. These data suggest that SUMOylation inhibition may lead to a double-punch effect against DMG by inhibiting tumorgenicity and stimulating an immune response. In further studies, we have shown that pharmacological inhibition of PAD activity in DMG decreases histone citrullination and restores histone methylation – considered the Achilles heel of DMG. This was accompanied by induction of reactive oxygen species and an unfolded protein response, resulting in DMG cell death in a dose dependent manner. In addition to fully characterising the epigenetic and global changes within DMG initiated through SUMOylation or PAD inhibition, we are currently performing preliminary drug efficacy studies in orthotopic DMG models, providing the necessary pre-clinical research pipeline towards an effective treatment for DMG. CONCLUSIONS Protein SUMOylation and citrullination are previously unrecognised therapeutic targets in DMG and potentially play key roles in tumour initiation and pathogenesis. Our data suggests a novel therapeutic approach for children with DMG.

Targeted inhibition of SUMOylation: treatment of tumors.

SUMOylation is a dynamic and reversible post-translational modification (PTM) of proteins involved in the regulation of biological processes such as protein homeostasis, DNA repair and cell cycle in normal and tumor cells. In particular, overexpression of SUMOylation components in tumor cells increases the activity of intracellular SUMOylation, protects target proteins against ubiquitination degradation and activation, promoting tumor cell proliferation and metastasis, providing immune evasion and increasing tolerance to chemotherapy and antitumor drugs. However, with the continuous research on SUMOylation and with the continued development of SUMOylation inhibitors, it has been found that tumor initiation and progression can be inhibited by blocking SUMOylation and/or in combination with drugs. SUMOylation is not a bad target when trying to treat tumor. This review introduces SUMOylation cycle pathway and summarizes the role of SUMOylation in tumor initiation and progression and SUMOylation inhibitors and their functions in tumors and provides a prospective view of SUMOylation as a new therapeutic target for tumors.

Repression of the SUMO-conjugating enzyme UBC9 is associated with lowered double minutes and reduced tumor progression

ABSTRACT Double minutes (DMs), extrachromosomal gene fragments found within certain tumors, have been noted to carry onco- and drug resistance genes contributing to tumor pathogenesis and progression. After screening for SUMO-related molecule expression within various tumor sample and cell line databases, we found that SUMO-conjugating enzyme UBC9 has been associated with genome instability and tumor cell DM counts, which was confirmed both in vitro and in vivo. Karyotyping determined DM counts post-UBC9 knockdown or SUMOylation inhibitor 2-D08, while RT-qPCR and Western blot were used to measure DM-carried gene expression in vitro. In vivo, fluorescence in situ hybridization (FISH) identified micronucleus (MN) expulsion. Western blot and immunofluorescence staining were then used to determine DNA damage extent, and a reporter plasmid system was constructed to detect changes in homologous recombination (HR) and non-homologous end joining (NHEJ) pathways. Our research has shown that UBC9 inhibition is able to attenuate DM formation and lower DM-carried gene expression, in turn reducing tumor growth and malignant phenotype, via MN efflux of DMs and lowering NHEJ activity to increase DNA damage. These findings thus reveal a relationship between heightened UBC9 activity, increased DM counts, and tumor progression, providing a potential approach for targeted therapies, via UBC9 inhibition.

MITF regulates the subcellular location of HIF1α through SUMOylation to promote the invasion and metastasis of daughter cells derived from polyploid giant cancer cells.

High concentrations of cobalt chloride (CoCl2) can induce the formation of polyploid giant cancer cells (PGCCs) in various tumors, which can produce daughter cells with strong proliferative, migratory and invasive abilities via asymmetric division. To study the role of hypoxia‑inducible factor (HIF) 1α in the formation of PGCCs, colon cancer cell lines Hct116 and LoVo were used as experimental subjects. Western blotting, nuclear and cytoplasmic protein extraction and immunocytochemical experiments were used to compare the changes in the expression and subcellular localization of HIF1α, microphthalmia‑associated transcription factor (MITF), protein inhibitor of activated STAT protein4 (PIAS4) and von Hippel‑Lindau disease tumor suppressor (VHL) after treatment with CoCl2. The SUMOylation of HIFα was verified by co‑immunoprecipitation assay. After inhibiting HIF1α SUMOylation, the changes in proliferation, migration and invasion abilities of Hct116 and LoVo were compared by plate colony formation, wound healing and Transwell migration and invasion. In addition, lysine sites that led to SUMOylation of HIF1α were identified through site mutation experiments. The results showed that CoCl2 can induce the formation of PGCCs with the expression level of HIF1α higher in treated cells than in control cells. HIF1α was primarily located in the cytoplasm of control cell. Following CoCl2 treatment, the subcellular localization of HIF1α was primarily in the nuclei of PGCCs with daughter cells (PDCs). After treatment with SUMOylation inhibitors, the nuclear HIF1α expression in PDCs decreased. Furthermore, their proliferation, migration and invasion abilities also decreased. After inhibiting the expression of MITF, the expression of HIF1α decreased. MITF can regulate HIF1α SUMOylation. Expression and subcellular localization of VHL and HIF1α did not change following PIAS4 knockdown. SUMOylation of HIF1α occurs at the amino acid sites K391 and K477 in PDCs. After mutation of the two sites, nuclear expression of HIF1α in PDCs was reduced, along with a significant reduction in the proliferation, migration and invasion abilities. In conclusion, the post‑translation modification regulated the subcellular location of HIF1α and the nuclear expression of HIF1α promoted the proliferation, migration and invasion abilities of PDCs. MITF could regulate the transcription and protein levels of HIF1α and participate in the regulation of HIF1α SUMOylation.

SUMOylation Inhibition Enhances Protein Transcription under CMV Promoter: A Lesson from a Study with the F508del-CFTR Mutant.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a selective anion channel expressed in the epithelium of various organs. The most frequent mutation is F508del. This mutation leads to a misfolded CFTR protein quickly degraded via ubiquitination in the endoplasmic reticulum. Although preventing ubiquitination stabilizes the protein, functionality is not restored due to impaired plasma membrane transport. However, inhibiting the ubiquitination process can improve the effectiveness of correctors which act as chemical chaperones, facilitating F508del CFTR trafficking to the plasma membrane. Previous studies indicate a crosstalk between SUMOylation and ubiquitination in the regulation of CFTR. In this study, we investigated the potential of inhibiting SUMOylation to increase the effects of correctors and enhance the rescue of the F508del mutant across various cell models. In the widely used CFBE41o-cell line expressing F508del-CFTR, inhibiting SUMOylation substantially boosted F508del expression, thereby increasing the efficacy of correctors. Interestingly, this outcome did not result from enhanced stability of the mutant channel, but rather from augmented cytomegalovirus (CMV) promoter-mediated gene expression of F508del-CFTR. Notably, CFTR regulated by endogenous promoters in multiple cell lines or patient cells was not influenced by SUMOylation inhibitors.

1,10-phenanthroline inhibits sumoylation and reveals that yeast SUMO modifications are highly transient

The steady-state levels of protein sumoylation depend on relative rates of conjugation and desumoylation. Whether SUMO modifications are generally long-lasting or short-lived is unknown. Here we show that treating budding yeast cultures with 1,10-phenanthroline abolishes most SUMO conjugations within one minute, without impacting ubiquitination, an analogous post-translational modification. 1,10-phenanthroline inhibits the formation of the E1~SUMO thioester intermediate, demonstrating that it targets the first step in the sumoylation pathway. SUMO conjugations are retained after treatment with 1,10-phenanthroline in yeast that express a defective form of the desumoylase Ulp1, indicating that Ulp1 is responsible for eliminating existing SUMO modifications almost instantly when de novo sumoylation is inhibited. This reveals that SUMO modifications are normally extremely transient because of continuous desumoylation by Ulp1. Supporting our findings, we demonstrate that sumoylation of two specific targets, Sko1 and Tfg1, virtually disappears within one minute of impairing de novo sumoylation. Altogether, we have identified an extremely rapid and potent inhibitor of sumoylation, and our work reveals that SUMO modifications are remarkably short-lived.

Dysregulated gene expression of SUMO machinery components induces the resistance to anti-PD-1 immunotherapy in lung cancer by upregulating the death of peripheral blood lymphocytes.

The majority of patients with lung cancer exhibit drug resistance after anti-PD-1 immunotherapy, leading to shortened patient survival time. Previous studies have suggested an association between epigenetic abnormalities such as methylation and clinical response to anti-PD-1 immunotherapy, while the role of SUMOylation in resistance to anti-PD-1 antibody immunotherapy is still unclear. Here, the mRNA expression of 15 SUMO machinery components in PBMC from lung cancer patients receiving anti-PD-1 immunotherapy were analyzed using real-time PCR. Base on the percentage change in mRNA levels, the relationship between the expression of SUMO machinery components and outcomes of anti-PD-1 immunotherapy, and the influencing factors of SUMOylation were evaluated. PBMC was treated with different concentrations of 2-D08 (a specific inhibitor of SUMOylation) in vitro, and analyzed the activation and the death rates of lymphocyte subsets by flow cytometry analysis. A predictive method, base on the gene expression of three SUMO machinery components (SUMO1, SUMO3 and UBE2I), were developed to distinguish non-responders to PD-1 inhibitors. Furthermore, the number of lymphocytes in peripheral blood significantly reduced in the dysregulated SUMOylation groups (the percentage change >100 or -50 ~ -100 groups). In vitro studies confirmed that lightly low SUMOylation level improved the activation status of T and NK lymphocytes, but extremely low SUMOylation level lead to the increased death rates of lymphocytes. Our findings implied that dysregulated gene expression of SUMO machinery components could induce the resistance of anti-PD-1 immunotherapy in lung cancer by upregulating the death of peripheral blood lymphocytes. These data might provide effective circulating biomarkers for predicting the efficacy of anti-PD-1 immunotherapy, and uncovered a novel regulatory mechanism of resistance to anti-PD-1 immunotherapy.

PTEN-mediated dephosphorylation of 53BP1 confers cellular resistance to DNA damage in cancer cells.

Homologous recombination (HR) repair for DNA double-strand breaks (DSBs) is critical for maintaining genome stability and conferring the resistance of tumor cells to chemotherapy. Nuclear PTEN which contains both phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and protein phosphatase plays a key role in HR repair, but the underlying mechanism remains largely elusive. We find that SUMOylated PTEN promotes HR repair but represses nonhomologous end joining (NHEJ) repair by directly dephosphorylating TP53-binding protein 1 (53BP1). During DNA damage responses (DDR), tumor suppressor ARF (p14ARF) was phosphorylated and then interacted efficiently with PTEN, thus promoting PTEN SUMOylation as an atypical SUMO E3 ligase. Interestingly, SUMOylated PTEN was subsequently recruited to the chromatin at DSB sites. This was because SUMO1 that was conjugated to PTEN was recognized and bound by the SUMO-interacting motif (SIM) of breast cancer type 1 susceptibility protein (BRCA1), which has been located to the core of 53BP1 foci on chromatin during S/G2 stage. Furthermore, these chromatin-loaded PTEN directly and specifically dephosphorylated phosphothreonine-543 (pT543) of 53BP1, resulting in the dissociation of the 53BP1 complex, which facilitated DNA end resection and ongoing HR repair. SUMOylation-site-mutated PTENK254R mice also showed decreased DNA damage repair in vivo. Blocking the PTEN SUMOylation pathway with either a SUMOylation inhibitor or a p14ARF(2-13) peptide sensitized tumor cells to chemotherapy. Our study therefore provides a new mechanistic understanding of PTEN in HR repair and clinical intervention of chemoresistant tumors.

SUMOylation inhibitors activate anti-tumor immunity by reshaping the immune microenvironment in a preclinical model of hepatocellular carcinoma.

High levels of heterogeneity and immunosuppression characterize the HCC immune microenvironment (TME). Unfortunately, the majority of hepatocellular carcinoma (HCC) patients do not benefit from immune checkpoint inhibitors (ICIs) therapy. New small molecule therapies for the treatment of HCC are the goal of our research. SUMOylation inhibitors (TAK-981 and ML-792) were evaluated for the treatment of preclinical mouse HCC models (including subcutaneous and orthotopic HCC models). We profile immune cell subsets from tumor samples after SUMOylation inhibitors treatment using single-cell RNA sequencing (scRNA-seq), mass cytometry (CyTOF), flow cytometry, and multiple immunofluorescences (mIF). We discover that SUMOylation is higher in HCC patient samples compared to normal liver tissue. TAK-981 and ML-792 decrease SUMOylation at nanomolar levels in HCC cells and also successfully reduced the tumor burden. Analysis combining scRNA-seq and CyTOF demonstrate that treatment with SUMOylation inhibitors reduces the exhausted CD8+T (Tex) cells while enhancing the cytotoxic NK cells, M1 macrophages and cytotoxic T lymphocytes (CTL) in preclinical mouse HCC model. Furthermore, SUMOylation inhibitors have the potential to activate innate immune signals from CD8+T, NK and macrophages while promoting TNFα and IL-17 secretion. Most notably, SUMOylation inhibitors can directly alter the TME by adjusting the abundance of intestinal microbiota, thereby restoring anti-tumor immunity in HCC models. This preclinical study suggests that SUMO signaling inhibitors may be beneficial for the treatment of HCC.

Nuclear Shuttling of IGF1R/INSR in Alzheimer’s disease and its corresponding role in regulation of neurodegeneration

AbstractBackgroundAlzheimer’s disease (AD) has been long associated with ageing and Insulin dysregulation. The receptors and ligands of the Insulin family including Insulin, IGF1, INSR, and IGF1R mainly has been explored over the years. However, their differential distribution in terms of compartmentalization and trafficking requires investigation.MethodWe started this study with SHSY5Y cells and checked for differential distribution in nucleus and cytoplasm in case of AD model. We repeated the study in N2A cells. To understand the nuclear shuttling better, we extended our study in differentiated SHSY5Y (into the Acetylcholinergic lineage) and exploited super resolution microscopy for imaging and trafficking. We fractionated the cellular compartments for biochemical evidence. To ascertain the partners and forms of protein that shuttles, antibody based assays were performed. For validation of this phenomena as of uniform occurrence, mice brain tissues and human primary neurons were utilized for imaging studies.ResultLigand independent shuttling into the nucleus occurs in case of AD models in presence of amyloid beta (1‐42). There is a 6.4 fold increase in phosphorylation in cytosolic pIGF1R/INSR in case of AD and a 2.5 fold increase in nuclear fractions. The occurrence correlated with the increase in Cyclin D1 production as suggested by ChIP studies. Upon treatment with Sumoylation inhibitor which blocks IGF1R transit into the nucleus, Cyclin D1 levels lowered validating transcriptional control of Cyclin D1 by IGF1R in AD scenario. Thus cell cycle can be directly influenced through interaction of IGF1R with binding partners LEF and TCF in AD.ConclusionCanonical ligand independent shuttling of phosphorylated IGF1R/INSR influences the cell cycle in AD thus triggering cell cycle through a direct control mechanism.

Metabolic Reprograming of Exhausted Intratumoral CD8+ T-Cell Underlies Anti-Tumor Activity of Sumoylation Inhibitors in Large B Cell Lymphoma

Immune checkpoint inhibitors (ICI) targeting PD1 have revolutionized our approach to solid tumors and Hodgkin lymphoma, but they have not been effective in treating Large B Cell Lymphoma (LBCL). In prior work, we used single cell spatial profiling of human tumors to show that exhausted CD8 tumor and TREG populations were associated with PD1 resistance in LBCL (Colombo et al. Blood Adv 2022). Here we show, using a syngeneic model of LBCL, that PD1 resistance is associated with lower CD8+ T-cell basal metabolic rate (BMR) in tumor infiltrating lymphocytes (TIL), and that treatment with the covalent SUMOylation inhibitor (SB-4826) increases the metabolic rate of TIL by metabolic reprograming. Single cell metabolic profiling of human LBCL is on-going and will be presented as well. In summary these studies demonstrate that metabolic dysfunction of T cells in LBCL underlie resistance to PD1 checkpoint inhibitors and that this can be overcome by treatment with SUMOlyation inhibitors. Direct measurement of intra-tumoral metabolic state is difficult to perform on clinical samples, however large tumor size is hypothesized to be a surrogate for increased metabolic derangement. As tumors expand and become more metabolically active themselves, infiltrating immune cells experience decreased O2 tension as well as nutrient deprivation, ultimately leading to decreased anti-tumor immune responses. In support of this idea, it has been observed that high metabolic tumor volume at baseline predicts survival in LBCL patients, independent of their initial response to therapy. We have shown that mice with established (>125mm 3) A20 tumors are resistant to anti-PD1 therapy. However, A20 bearing mice treated ~5 days post implantation are responsive to anti-PD1 therapy. To understand these findings, we examined sensitive (small) and resistant (large) A20 tumors by flow cytometry analysis and found that resistant tumors had lower relative numbers of T cells, lower absolute numbers of CD8+ T cells, increased absolute numbers of TREG and a strikingly decreased CD8/TREG ratio. Additionally, the resistant tumors had increased absolute numbers of pre-exhausted and exhausted CD8+ T cells as well as decreased BMR. Small ubiquitin-like modifiers (SUMO) proteins regulate a variety of cellular processes in tumor and immune cells and inhibitors of SUMOylation have shown anti-tumor activity in a variety of preclinical tumor models with potential synergy with checkpoint inhibitors targeting PD-1 (Cannon et al. Cancer Res, 2023). We demonstrate that a dose of 100mg/kg of SB-4826 led to a complete response in all mice treated (Fig. 1A) and we also show that this effect is dependent on CD8+ T-cells and elicits a long term memory response, with 0 of 15 mice in remission developing tumor upon rechallenge. A low dose (25mg/kg) of SB-4826 failed to elicit a response in any of the mice treated while anti-PD1 therapy elicited a response in 1/6 mice (Fig. 1A). Combination of 25mg/kg SB-4826 with anti-PD1 therapy demonstrated synergism with a response in 3/6 mice (Fig. 1A). We also demonstrate that global SUMOylation levels are higher in PD1+ TIM3+ CD8+ TILs compared to PD1- TIM3- CD8+ T-cells, suggesting that SUMOylation may be acting as a negative regulator of T-cell function. Using SCENITH (Arguello et al. Cell Metabolism 2020) for single cell metabolic studies, we demonstrate that SB-4826 increases the BMR (Fig. 1B), mitochondrial dependency and proliferative capacity of tumor infiltrating PD1+ TIM3+ CD8+ T-cells (TILs). While both SB-4826 and PD1 inhibition led to increased CD8+ TIL numbers, SB-4826 led to increased BMR in CD8+ TILs while PD1 inhibition did not affect CD8+ TILs BMR. Additionally, PD1 inhibition significantly decreased CD8+ TILs mitochondrial dependency for metabolism, while SB-4826 increased mitochondrial dependency (Fig. 1B). Additionally, we recapitulate these metabolic enhancements in an in vitro system of T-cell activation, further confirming a T-cell intrinsic effect following SB-4826. Mechanistically, we found that SB-4826 led to increased HIF-1a levels in both CD4+ and CD8+ cells following TCR activation, likely through stabilization of HIF-1a due to decreased SUMOylation-dependent turnover (Cheng J et al. Cell 2007). We hypothesize that metabolic reprograming of CD8 T-cells through increased HIF-1a is the basis for synergy between SUMOylation inhibition and immune checkpoint inhibitors in LBCL.

Sumoylation in astrocytes induces changes in the proteome of the derived small extracellular vesicles which change protein synthesis and dendrite morphology in target neurons

Emerging evidence highlights the relevance of the protein post-translational modification by SUMO (Small Ubiquitin-like Modifier) in the central nervous system for modulating cognition and plasticity in health and disease. In these processes, astrocyte-to-neuron crosstalk mediated by extracellular vesicles (EVs) plays a yet poorly understood role. Small EVs (sEVs), including microvesicles and exosomes, contain a molecular cargo of lipids, proteins, and nucleic acids that define their biological effect on target cells. Here, we investigated whether SUMOylation globally impacts the sEV protein cargo. For this, sEVs were isolated from primary cultures of astrocytes by ultracentrifugation or using a commercial sEV isolation kit. SUMO levels were regulated: 1) via plasmids that over-express SUMO, or 2) via experimental conditions that increase SUMOylation, i.e., by using the stress hormone corticosterone, or 3) via the SUMOylation inhibitor 2-D08 (2′,3′,4′-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one). Corticosterone and 2-D08 had opposing effects on the number of sEVs and on their protein cargo. Proteomic analysis showed that increased SUMOylation in corticosterone-treated or plasmid-transfected astrocytes increased the presence of proteins related to cell division, transcription, and protein translation in the derived sEVs. When sEVs derived from corticosterone-treated astrocytes were transferred to neurons to assess their impact on protein synthesis using the fluorescence non-canonical amino acid tagging assay (FUNCAT), we detected an increase in protein synthesis, while sEVs from 2-D08-treated astrocytes had no effect. Our results show that SUMO conjugation plays an important role in the modulation of the proteome of astrocyte-derived sEVs with a potential functional impact on neurons.