Neddylation Inhibitor Research Articles

Neddylation is critical for cardiomyocyte maturation by promoting perinatal metabolic transition

The heart undergoes significant structural, metabolic, gene expression and functional alterations during the perinatal to postnatal transition. Although recent studies have delineated several novel epigenetic and transcriptional regulators of cardiac maturation, post‐transcriptional mechanisms regulating cardiomyocyte (CM) maturation remain poorly defined. We hypothesized that neddylation, a protein modification that conjugates a small ubiquitin‐like protein NEDD8 to protein substrates via an E1‐E2‐E3 cascade, plays a critical role in driving cardiac maturation program. Taking advantage of an AAV9‐cTnT‐Cre system and the mTmG reporter, we achieved CM‐specific, mosaic knockout of NAE1, a critical neddylation E1 enzyme, in postnatal hearts. Mice with high levels (>90%) of NAE1 deletion in CMs exhibited a cardiomyopathy as evidenced by significant chamber dilatation and greatly compromised cardiac contractility. While mice with modest levels (30%) of NAE1 deletion in CMs had preserved cardiac function, inhibition of neddylation caused severe disruption of transverse tubules (T‐tubules) network and a disturbance in binucleation and cell enlargement in NAE1‐null CMs, as well as upregulation of fetal genes and downregulation of their adult forms, indicating a key, cell‐autonomous role for neddylation in CM maturation. Transcriptome analysis revealed an upregulation of glycolytic genes and a downregulation of oxidative metabolic genes in NAE1‐deficient hearts. Biochemical analyses confirmed defects in fatty acid oxidative metabolism and mitochondrial dysfunction in NAE1‐deficient hearts and neonatal CMs treated with a neddylation inhibitor. Mechanistically, we found that HIF1α, a transcription factor known to promote glycolysis and suppress oxidative metabolism, is a putative NEDD8 target. Inhibition of neddylation stabilized HIF1α and led to abnormal accumulation of HIF1α in NAE1‐deficient hearts and neonatal CMs treated with neddylation inhibitor, which may contribute to the observed metabolic dysfunction. Together, our findings suggest that neddylation serves as a novel post‐translational mechanism regulating CM maturation through sustaining the glycolytic to oxidative metabolic switch in postnatal hearts.

Hepatic Neddylation Deficiency Triggers Fatal Liver Injury via Inducing NF‐κB‐inducing Kinase and Oxidative Stress

The conjugation of NEDD8 to target proteins, termed neddylation is aberrant in many pathological diseases. Its relevance to liver function and failure remains poorly understood. Here, we first identified dysregulated expression of neddylation enzymes (e.g. NAE1, a regulatory subunit of the only NEDD8 E1 enzyme) in human HBV‐induced acute liver failure (ALF). Embryonic–onset deletion of NAE1 in hepatocytes led to severe hepatomegaly, formation of numerous cysts, inflammation, and fibrosis, which ultimately resulted in ALF in mice. Adult–onset hepatic deletion of NAE1 caused ALF with 100% mortality within 30 days post deletion. Mechanistically, hepatic neddylation deficiency triggers glutathione deficiency, oxidative stress, mitochondrial dysfunction, and reprogramming of mature hepatocytes to fetal‐like cells, potentiating liver injury. Importantly, we identified NF‐κB‐inducing kinase (NIK), a stress kinase aberrantly activated in chronic and acute liver diseases, is a novel neddylation substrate. Neddylation of NIK promotes its ubiquitination and degradation; and inhibition of neddylation caused NIK overactivation, accentuating hepatocyte damage. Notably, oral N‐acetylcysteine (NAC) administration attenuated lethal liver injury caused by hepatic NAE1 deletion, with improved liver function and mortality. Therefore, hepatic neddylation is essential in maintaining postnatal and adult liver homeostasis, and the novel neddylation targets/pathways may endow us with new insights into therapeutically intervening ALF progression.

Pharmacological Inhibition of Cullin Neddylation Improves Hepatic Insulin Sensitivity

Background and significanceHepatic insulin resistance is a hallmark feature of non‐alcoholic fatty liver disease (NAFLD) and type‐2 diabetes (T2D) and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress‐sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin‐RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin‐like protein called Nedd8 to a cullin scaffold.AimTo investigate the role of CRLs in regulating hepatic insulin signaling in NAFLD.MethodsNeddylation inhibitor MLN4924 was used to study its effect on insulin sensitivity in liver cells. Hyperinsulinemic‐euglycemic clamp was used to assess MNL4924 effect on insulin sensitivity in vivo. siRNA‐mediated screening was used to identify the Cullin members that mediates IRS protein turnover in liver cells. Co‐immunoprecipitation was performed to study Cullin and IRS interaction. Glucose homeostasis and insulin sensitivity were studied in mice with AAV‐mediated liver specific knockdown of Cul1 or Cul3.ResultsNeddylation inhibition delays CRL‐mediated IRS protein turnover to prolong insulin action in cultured liver cells. Consistently, acute MLN4924 treatment rapidly decreases hepatic glucose production without causing hypoglycemia in mice, while chronic MLN4924 treatment completely normalizes hyperglycemia in obese mice independent of obesity. Hyperinsulinemic‐euglycemic clamp study revealed that MLN4924 significantly increased insulin sensitivity and improved hepatic glucose output. In vitro siRNA based screening showed that knockdown of Cul1 or Cul3, but not other cullin family members, stabilized IRS protein and increased insulin sensitivity in AML12 cells. Consistently, Co‐immunoprecipitation demonstrated IRS interaction with Cul1 and Cul3. AAV‐mediated liver specific knockdown of Cul1 in mice improved insulin sensitivity, lowered fasting glucose, and prevented Western diet‐induced obesity and NAFLD. In contrast, AAV‐mediated liver Cul3 knockdown only decreased fasting glucose in chow‐fed mice but not Western diet‐fed mice.ConclusionThese findings suggest that pharmacological inhibition of cullin neddylation is a novel therapeutic strategy for hepatic insulin sensitization in NAFLD and T2D. This effect may be at least in part mediated by hepatic Cul1 and Cul3 neddylation.

Involvement of the neddylation modification system in Bombyx mori nucleopolyhedrovirus replication.

Neddylation is a posttranslational modification that is similar to ubiquitination, and involved in some critical biological processes, such as DNA repair, transcription regulation, and ubiquitin-proteasome pathway. Recently, it was found that neddylation inhibitor MLN4924 has potent antiviral activity against human viruses including herpes simplex virus (HSV)-1, HSV-2, and influenza viruses. Here, we report that MLN4924 could dramatically and dose-dependently inhibits the propagation, formation of budding virus (BV) and occlusion body (OB) of a lepidopteran virus-Bombyx mori nucleopolyhedrovirus (BmNPV), impaired OB assembly. In addition, the neddylation modification protein NEDD8 is colocalized with aggresome and autophagosome. Our findings suggest that inhibiting neddylation could be an antibaculovirus strategy and MLN4924 may be used as candidate drug for that purpose.

Mechanical stress shapes the cancer cell response to neddylation inhibition

BackgroundThe inhibition of neddylation by the preclinical drug MLN4924 represents a new strategy to combat cancer. However, despite being effective against hematologic malignancies, its success in solid tumors, where cell–cell and cell-ECM interactions play essential roles, remains elusive.MethodsHere, we studied the effects of MLN4924 on cell growth, migration and invasion in cultured prostate cancer cells and in disease-relevant prostate tumoroids. Using focused protein profiling, drug and RNAi screening, we analyzed cellular pathways activated by neddylation inhibition.ResultsWe show that mechanical stress induced by MLN4924 in prostate cancer cells significantly affects the therapeutic outcome. The latter depends on the cell type and involves distinct Rho isoforms. In LNCaP and VCaP cells, the stimulation of RhoA and RhoB by MLN4924 markedly upregulates the level of tight junction proteins at cell–cell contacts, which augments the mechanical strain induced by Rho signaling. This “tight junction stress response” (TJSR) causes the collapse of cell monolayers and a characteristic rupture of cancer spheroids. Notably, TJSR is a major cause of drug-induced apoptosis in these cells. On the other hand, in PC3 cells that underwent partial epithelial-to-mesenchymal transition (EMT), the stimulation of RhoC induces an adverse effect by promoting amoeboid cell scattering and invasion. We identified complementary targets and drugs that allow for the induction of TJSR without stimulating RhoC.ConclusionsOur finding that MLN4924 acts as a mechanotherapeutic opens new ways to improve the efficacy of neddylation inhibition as an anticancer approach.Graphical

A review on cullin neddylation and strategies to identify its inhibitors for cancer therapy.

The cullin-RING E3 ligases (CRLs) are the biggest components of the E3 ubiquitin ligase protein family, and they represent an essential role in various diseases that occur because of abnormal activation, particularly in tumors development. Regulation of CRLs needs neddylation, a post-translational modification involving an enzymatic cascade that transfers small, ubiquitin-like NEDD8 protein to CRLs. Many previous studies have confirmed neddylation as an enticing target for anticancer drug discoveries, and few recent studies have also found a significant increase in advancement in protein neddylation, including preclinical and clinical target validation to discover the neddylation inhibitor compound. In the present review, we first presented briefly the essence of CRLs' neddylation and its control, systematic analysis of CRLs, followed by the description of a few recorded chemical inhibitors of CRLs neddylation enzymes with recent examples of preclinical and clinical targets. We have also listed various structure-based pointing of protein-protein dealings in the CRLs' neddylation reaction, and last, the methods available to discover new inhibitors of neddylation are elaborated. This review will offer a concentrated, up-to-date, and detailed description of the discovery of neddylation inhibitors.

PTEN loss drives resistance to the neddylation inhibitor MLN4924 in glioblastoma and can be overcome with TOP2A inhibitors.

Neddylation inhibition, affecting posttranslational protein function and turnover, is a promising therapeutic approach to cancer. We report vulnerability to MLN4924 or pevonedistat (a neddylation inhibitor) in a subset of glioblastoma (GBM) preclinical models and identify biomarkers, mechanisms, and signatures of differential response. GBM sequencing data were queried for genes associated with MLN4924 response status; candidates were validated by molecular techniques. Time-course transcriptomics and proteomics revealed processes implicated in MLN4924 response. Vulnerability to MLN4924 is associated with elevated S-phase populations, re-replication, and DNA damage. Transcriptomics and shotgun proteomics depict PTEN signaling, DNA replication, and chromatin instability pathways as significant differentiators between sensitive and resistant models. Loss of PTEN and its nuclear functions is associated with resistance to MLN4924. Time-course proteomics identified elevated TOP2A in resistant models through treatment. TOP2A inhibitors combined with MLN4924 prove synergistic. We show that PTEN status serves as both a novel biomarker for MLN4924 response in GBM and reveals a vulnerability to TOP2A inhibitors in combination with MLN4924.

Mouse Adenovirus Type 1 E4orf6 Induces PKR Degradation.

Protein kinase R (PKR) is a cellular kinase involved in the antiviral response. The inactivation or inhibition of this protein is a conserved activity in DNA and RNA virus infections. In contrast to human adenovirus type 5, mouse adenovirus type 1 (MAV-1) inhibits PKR activity through proteasome-dependent degradation. However, the molecular mechanism by which this process takes place is not fully understood. We investigated whether ubiquitination, MAV-1 early region 1B 55k (E1B 55k), and early region 4 orf6 (E4orf6) play a role in PKR degradation in MAV-1 infection, because the enzyme 3 (E3) ubiquitin ligase activity with these viral proteins is conserved among the Adenoviridae family. We provide evidence that E4orf6 is sufficient to induce mouse PKR degradation and that proteasome pathway inhibition blocks PKR degradation. Inhibition of neddylation of cullin, a component of E3 ubiquitin ligase complex, blocked efficient PKR degradation in MAV-1-infected cells. Finally, we demonstrated that MAV-1 degradation of PKR is specific for mouse PKR. These results indicate that counteracting PKR is mechanistically different in two species of adenoviruses. IMPORTANCE Viruses have evolved to counteract the immune system to successfully replicate in the host. Downregulation of several antiviral proteins is important for productive viral infection. Protein kinase R (PKR) is an antiviral protein that belongs to the first line of defense of the host. Because PKR senses dsRNA and blocks the cellular translation process during viral infections, it is not surprising that many viruses counteract this antiviral activity. We previously reported PKR degradation during mouse adenovirus type 1 (MAV-1) infection; however, the molecular mechanism of this activity was not fully known. This work provides evidence about the MAV-1 protein that induces PKR degradation and expands knowledge about involvement of the proteasome pathway.

Neddylation is essential for β-catenin degradation in Wnt signaling pathway.

β-Catenin is a central component in the Wnt signaling pathway; its degradation has been tightly connected to ubiquitylation, but it is rarely examined by loss-of-function assays. Here we observe that endogenous β-catenin is not stabilized upon ubiquitylation depletion by a ubiquitylation inhibitor, TAK-243. We demonstrate that N-terminal phosphorylated β-catenin is quickly and strongly stabilized by a specific neddylation inhibitor, MLN4924, in all examined cell types, and that β-catenin and TCF4 interaction is strongly enhanced by inhibition of neddylation but not ubiquitylation. We also confirm that the E3 ligase β-TrCP2, but not β-TrCP1, is associated with neddylation and destruction of β-catenin. GSK3β and adenomatous polyposis coli (APC) are not required for β-catenin neddylation but essential for its subsequent degradation. Our findings not only clarify the process of β-catenin modification and degradation in the Wnt signaling pathway but also highlight the importance of reassessing previously identified ubiquitylation substrates.

The Cellular and Developmental Roles of Cullins, Neddylation, and the COP9 Signalosome in Dictyostelium discoideum.

Cullins (CULs) are a core component of cullin-RING E3 ubiquitin ligases (CRLs), which regulate the degradation, function, and subcellular trafficking of proteins. CULs are post-translationally regulated through neddylation, a process that conjugates the ubiquitin-like modifier protein neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target cullins, as well as non-cullin proteins. Counteracting neddylation is the deneddylase, COP9 signalosome (CSN), which removes NEDD8 from target proteins. Recent comparative genomics studies revealed that CRLs and the CSN are highly conserved in Amoebozoa. A well-studied representative of Amoebozoa, the social amoeba Dictyostelium discoideum, has been used for close to 100 years as a model organism for studying conserved cellular and developmental processes owing to its unique life cycle comprised of unicellular and multicellular phases. The organism is also recognized as an exceptional model system for studying cellular processes impacted by human diseases, including but not limited to, cancer and neurodegeneration. Recent work shows that the neddylation inhibitor, MLN4924 (Pevonedistat), inhibits growth and multicellular development in D. discoideum, which supports previous work that revealed the cullin interactome in D. discoideum and the roles of cullins and the CSN in regulating cellular and developmental processes during the D. discoideum life cycle. Here, we review the roles of cullins, neddylation, and the CSN in D. discoideum to guide future work on using this biomedical model system to further explore the evolutionarily conserved functions of cullins and neddylation.

Targeting NAE1-mediated protein hyper-NEDDylation halts cholangiocarcinogenesis and impacts on tumor-stroma crosstalk in experimental models

Cholangiocarcinoma (CCA) comprises a heterogeneous group of malignant tumors associated with dismal prognosis. Alterations in post-translational modifications (PTMs), including NEDDylation, result in abnormal protein dynamics, cell disturbances and disease. Herein, we investigate the role of NEDDylation in CCA development and progression. Levels and functions of NEDDylation, together with response to pevonedistat (NEDDylation inhibitor) or CRISPR/Cas9 against NAE1 were evaluated invitro, invivo and/or in patients with CCA. The development of preneoplastic lesions in Nae1+/- mice was investigated using an oncogene-driven CCA model. The impact of NEDDylation in CCA cells on tumor-stroma crosstalk was assessed using CCA-derived cancer-associated fibroblasts (CAFs). Proteomic analyses were carried out by mass-spectrometry. The NEDDylation machinery was found overexpressed and overactivated in human CCA cells and tumors. Most NEDDylated proteins found upregulated in CCA cells, after NEDD8-immunoprecipitation and further proteomics, participate in the cell cycle, proliferation or survival. Genetic (CRISPR/Cas9-NAE1) and pharmacological (pevonedistat) inhibition of NEDDylation reduced CCA cell proliferation and impeded colony formation invitro. NEDDylation depletion (pevonedistat or Nae1+/- mice) halted tumorigenesis in subcutaneous, orthotopic, and oncogene-driven models of CCA invivo. Moreover, pevonedistat potentiated chemotherapy-induced cell death in CCA cells invitro. Mechanistically, impaired NEDDylation triggered the accumulation of both cullin RING ligase and NEDD8 substrates, inducing DNA damage and cell cycle arrest. Furthermore, impaired NEDDylation in CCA cells reduced the secretion of proteins involved in fibroblast activation, angiogenesis, and oncogenic pathways, ultimately hampering CAF proliferation and migration. Aberrant protein NEDDylation contributes to cholangiocarcinogenesis by promoting cell survival and proliferation. Moreover, NEDDylation impacts the CCA-stroma crosstalk. Inhibition of NEDDylation with pevonedistat may represent a potential therapeutic strategy for patients with CCA. Little is known about the role of post-translational modifications of proteins in cholangiocarcinoma development and progression. Herein, we show that protein NEDDylation is upregulated and hyperactivated in cholangiocarcinoma, promoting tumor growth. Pharmacological inhibition of NEDDylation halts cholangiocarcinogenesis and could be an effective therapeutic strategy to tackle these tumors.

SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma

BackgroundThe combination of pemetrexed and cisplatin remains the reference first-line systemic therapy for malignant pleural mesothelioma (MPM). Its activity is moderate because of tumor aggressiveness, immune-suppressive environment and resistance to chemotherapy-induced immunogenic cell death (ICD). Preliminary and limited findings suggest that MPM cells have deregulated ubiquitination and proteasome activities, although proteasome inhibitors achieved disappointing clinical results.MethodsHere, we investigated the role of the E3-ubiquitin ligase SKP/Cullin/F-box (SCF) complex in cell cycle progression, endoplasmic reticulum (ER)/proteostatic stress and ICD in MPM, and the therapeutic potential of the neddylation/SCF complex inhibitor MLN4924/Pevonedistat.ResultsIn patient-derived MPM cultures and syngenic murine models, MLN4924 and cisplatin showed anti-tumor effects, regardless of MPM histotype and BAP1 mutational status, increasing DNA damage, inducing S- and G2/M-cell cycle arrest, and apoptosis. Mechanistically, by interfering with the neddylation of cullin-1 and ubiquitin-conjugating enzyme UBE2M, MLN4924 blocks the SCF complex activity and triggers an ER stress-dependent ICD, which activated anti-MPM CD8+T-lymphocytes. The SKP2 component of SCF complex was identified as the main driver of sensitivity to MLN4924 and resistance to cisplatin. These findings were confirmed in a retrospective MPM patient series, where SKP2 high levels were associated with a worse response to platinum-based therapy and inferior survival.ConclusionsWe suggest that the combination of neddylation inhibitors and cisplatin could be worth of further investigation in the clinical setting for MPM unresponsive to cisplatin. We also propose SKP2 as a new stratification marker to determine the sensitivity to cisplatin and drugs interfering with ubiquitination/proteasome systems in MPM.

Deneddylation of PML/RARα reconstructs functional PML nuclear bodies via orchestrating phase separation to eradicate APL.

Acute promyelocytic leukemia (APL) is driven by the oncoprotein PML/RARα, which destroys the architecture of PML nuclear bodies (NBs). PML NBs are critical to tumor suppression, and their disruption mediated by PML/RARα accelerates APL pathogenesis. However, the mechanisms of PML NB disruption remain elusive. Here, we reveal that the failure of NB assembly in APL results from neddylation-induced aberrant phase separation of PML/RARα. Mechanistically, PML/RARα is neddylated in the RARα moiety, and this neddylation enhances its DNA-binding ability and further impedes the phase separation of the PML moiety, consequently disrupting PML NB construction. Accordingly, deneddylation of PML/RARα restores its phase separation process to reconstruct functional NBs and activates RARα signaling, thereby suppressing PML/RARα-driven leukemogenesis. Pharmacological inhibition of neddylation by MLN4924 eradicates APL cells both in vitro and in vivo. Our work elucidates the neddylation-destroyed phase separation mechanism for PML/RARα-driven NB disruption and highlights targeting neddylation for APL eradication.

Cullin neddylation inhibitor attenuates hyperglycemia by enhancing hepatic insulin signaling through insulin receptor substrate stabilization

Hepatic insulin resistance is a hallmark feature of nonalcoholic fatty liver disease and type-2 diabetes and significantly contributes to systemic insulin resistance. Abnormal activation of nutrient and stress-sensing kinases leads to serine/threonine phosphorylation of insulin receptor substrate (IRS) and subsequent IRS proteasome degradation, which is a key underlying cause of hepatic insulin resistance. Recently, members of the cullin-RING E3 ligases (CRLs) have emerged as mediators of IRS protein turnover, but the pathophysiological roles and therapeutic implications of this cellular signaling regulation is largely unknown. CRLs are activated upon cullin neddylation, a process of covalent conjugation of a ubiquitin-like protein called Nedd8 to a cullin scaffold. Here, we report that pharmacological inhibition of cullin neddylation by MLN4924 (Pevonedistat) rapidly decreases hepatic glucose production and attenuates hyperglycemia in mice. Mechanistically, neddylation inhibition delays CRL-mediated IRS protein turnover to prolong insulin action in hepatocytes. In vitro knockdown of either cullin 1 or cullin 3, but not other cullin members, attenuates insulin-induced IRS protein degradation and enhances cellular insulin signaling activation. In contrast, invivo knockdown of liver cullin 3, but not cullin 1, stabilizes hepatic IRS and decreases blood glucose, which recapitulates the effect of MLN4924 treatment. In summary, these findings suggest that pharmacological inhibition of cullin neddylation represents a therapeutic approach for improving hepatic insulin signaling and lowering blood glucose.

The NEDD8-activating enzyme inhibitor MLN4924 reduces ischemic brain injury in mice

Blood-brain barrier (BBB) breakdown and inflammation occurring at the BBB have a key, mainly a deleterious role in the pathophysiology of ischemic stroke. Neddylation is a ubiquitylation-like pathway that is critical in various cellular functions by conjugating neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8) to target proteins. However, the roles of neddylation pathway in ischemic stroke remain elusive. Here, we report that NEDD8 conjugation increased during acute phase after ischemic stroke and was present in intravascular and intraparenchymal neutrophils. Inhibition of neddylation by MLN4924, also known as pevonedistat, inactivated cullin-RING E3 ligase (CRL), and reduced brain infarction and improved functional outcomes. MLN4924 treatment induced the accumulation of the CRL substrate neurofibromatosis 1 (NF1). By using virus-mediated NF1 silencing, we show that NF1 knockdown abolished MLN4924-dependent inhibition of neutrophil trafficking. These effects were mediated through activation of endothelial P-selectin and intercellular adhesion molecule-1 (ICAM-1), and blocking antibodies against P-selectin or anti-ICAM-1 antibodies reversed NF1 silencing-induced increase in neutrophil infiltration in MLN4924-treated mice. Furthermore, we found that NF1 silencing blocked MLN4924-afforded BBB protection and neuroprotection through activation of protein kinase C δ (PKCδ), myristoylated alanine-rich C-kinase substrate (MARCKS), and myosin light chain (MLC) in cerebral microvessels after ischemic stroke, and treatment of mice with the PKCδ inhibitor rottlerin reduced this increased BBB permeability. Our study demonstrated that increased neddylation promoted neutrophil trafficking and thus exacerbated injury of the BBB and stroke outcomes. We suggest that the neddylation inhibition may be beneficial in ischemic stroke.

Abstract P051: Identification of an orally bioavailable dual Cyclin K glue degrader - CDK12/13 inhibitor

Abstract CDK12 and CDK13 regulate gene expression through translation and splicing of mRNA. Impaired activity of CDK12 has been shown to reduce the expression of a range of genes involved in DNA homologous repair leading to the induction of a ‘BRCAness’ phenotype in cells. CDK12 inhibitors have the potential in many different indications, including overcoming emergent resistance to PARP inhibition, enhancing the efficacy of PARP inhibition in patients with BRCA1/2 wt status [1] and extending the efficacy of immune checkpoint inhibition through immunogenic cell death [2]. Recently, certain classes of CDK12 inhibitors have also demonstrated ‘glue degrader’ properties [1]: causing proteosomal degradation of the obligate co-factor for CDK12 and 13, Cyclin K. Such glue-degraders have the capability of enhancing cellular potency and specificity over classical kinase inhibitors. Taking as a starting point the selective CDK7 inhibitor samuraciclib, we have taken a rational design approach to develop inhibitors that preferentially inhibit CDK12. By utilising differences in the active site between CDK7 and CDK12 we were able to increase potency against CDK12 in biochemical assays by > 100 fold, with no significant inhibition of kinases outside the CDK class. Cellular potency of these molecules was evaluated in the Ewing’s sarcoma cell line A673, which is known to be sensitive to CDK12 inhibition [4]. Remarkably the potency of the molecules to induce cell death was greater than their biochemical potency against CDK12 by > 10 fold. Analysis of Cyclin K levels in cells demonstrated that the molecules lead to Cyclin K degradation. Furthermore, cellular potency was reduced by the presence of the neddylation inhibitor MLN4924 confirming that the compounds enhanced potency was mediated by glue degradation of Cyclin K. The molecules were found to cause potent inhibition of DNA homologous repair genes including BRCA1, FANCF and ERCC4 and lead to the accumulation of DNA strand breaks as assessed by the marker γ-H2AX. In the BRCA wt ovarian cancer cell line, OV-90, strong synergy was observed in the ability to induce cell death with a range of PARP inhibitors. In A673 tumour bearing mice, dosing with the compounds was found to cause significant reduction in the levels of cyclin K in tumours, which was sustained for 24 hr. Additionally, assessment of CDK12 protein in these tumours also showed a reduction, suggesting greater turnover of CDK12 protein in the absence of Cyclin K. Repeat dose studies at doses that lead to Cyclin K degradation in tumours demonstrated that the compounds were well tolerated, with no weight loss in mice. Two molecules, CT7439 and CT7510, were found to have oral bioavailability in mice that indicate that they would be suitable for once-daily oral administration in humans and are being progressed through regulatory toxicity studies. [1] Johnson, et al, (2016) Cell Rep. 17, 2367 [2] Li, et al, (2020) Cancer Lett. 495, 12 [3] Słabicki, et al, (2020) Nature 585, 293 [4] Iniguez, et al, (2018) Cancer Cell 33, 202 Citation Format: Edward K. Ainscow, Adrian Campbell, Michael Cripps, Robert Workman, Stuart Thomson, Kam Chohan, Damien Crepin, Mahiro Sunose, Jamie Patient, Jane Kendrew, Ash Bahl. Identification of an orally bioavailable dual Cyclin K glue degrader - CDK12/13 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P051.

Abstract 12880: Neddylation Regulates Murine Cardiomyocyte Maturation

From birth to adulthood cardiomyocytes (CMs) undergo a complex maturation process characterized by drastic changes in cell size, ultrastructure, metabolism and gene expression. The molecular mechanisms regulating CM maturation remain poorly defined. Neddylation is a novel post-translational modification that covalently attaches a small ubiquitin-like protein, NEDD8, to target proteins. We found that deletion of NAE1, a critical NEDD8 E1 enzyme that triggers neddylation, in embryonic and perinatal mouse hearts via Nkx2.5 Cre and aMHC Cre , respectively, led to myocardial hypoplasia, heart failure and early lethality. The severe cardiac phenotypes are accompanied by disorganized sarcomere structure and defective fetal-to-adult gene transition, suggesting a possible role for neddylation in CM maturation. To rule out the confounding effect of secondary changes caused by perturbed CM differentiation and cardiac stress response in the mutant hearts, we used AAV9-cTnT-Cre to achieve mosaic knockout of NAE1 in postnatal hearts. High-dose AAV9 deleted NAE1 in 80% CMs and caused heart failure, whereas low-dose AVV9 deleted NAE1 in 30% CMs and preserved heart function. In the context of preserved cardiac function, CMs lacking NAE1 showed severe disrupted transverse tubules (T-tubules) network, which was further exacerbated in hearts with modest dysfunction. In contrast, T-tubule morphology was largely maintained in CMs with intact NAE1 expression from low-dose AAV9 infected hearts, as well as in CMs with deletion of one allele of NAE1, indicating a key, cell-autonomous role of neddylation in T-tubule maturation. Biochemical and histological analyses further revealed dysregulated metabolic and sarcomeric gene expression and disrupted sarcomere ultrastructure in high-dose AAV9 infected hearts, supporting defective CM maturation. In vitro, pharmacological inhibition of neddylation also impaired myofibril assembly in isolated neonatal and adult CMs. Together, our data reveal an important role of neddylation in CM maturation in the developing heart, warranting further investigations of detailed posttranslational mechanisms underlying CM maturation.

CSIG-14. PTEN AS A SIGNATURE OF VULNERABILITY OF GBM TO NEDDYLATION INHIBITION

Abstract Neddylation is a specific pathway within the ubiquitin/proteasome system that is overactive in GBM, and whose upregulation has been associated with glioma progression and worse survival. Pevonedistat (MLN4924) is a first-in-class small-molecule neddylation inhibitor shown to inhibit growth of GBM cells by impacting protein degradation in culture and orthotopic xenografts. However, the determinants of vulnerability are not fully understood. Because the molecular heterogeneity within and across GBM patients obscures therapeutic targets and obfuscates signals of efficacy in clinical trials, we pursue the use of molecular “signatures of vulnerability” to targeted agents in subsets of preclinical models. Selective vulnerability to pevonedistat was shown in a subset of GBM; notably, models with mutations or copy number deletions of PTEN are associated with de novo resistance to pevonedistat. Time-course studies of sensitive and non-sensitive GBM cells using transcriptomics and proteomics/phosphoproteomics uncovered additional determinants of response to pevonedistat. Our results demonstrate that in GBM, resistance to pevonedistat is driven by reduced PTEN-chromatin binding (loss-of-function or lower expression) that is also independent of PTEN’s lipid phosphatase activity (i.e., PI3K/AKT signaling). Across 25 glioma cell lines, we found that PTEN signaling, DNA replication, and chromatin instability pathways are the most significant differentiators between pevonedistat sensitive vs. non-sensitive models. In GBM models with modest to low sensitivity to pevonedistat, TOP2A expression was elevated. Combination treatment with the TOP2A inhibitor, etoposide, proved synergistic with pevonedistat. We report that PTEN status both serves as a novel biomarker for GBM sensitivity to pevonedistat and reveals a synergistic vulnerability to TOP2A inhibitors in combination with pevonedistat. Paired use of GBM PDX models of varying sensitivity with drug development testing allows the advancement of a promising agent as well as a patient-enrollment “signature of vulnerability” likely to increase the likelihood of demonstrating therapeutic efficacy in early stage clinical trials.

Single Cell Transcriptomics Revealed Molecular Alterations in AML Cell Clusters Relevant to Refractory Disease at Relapse

Introduction: Drug resistance at relapse is a major cause of mortality in AML. Previous genomic profiling of AML patient samples revealed that in many cases the mutational profile did not change between diagnosis and relapse (Parkin et al., Blood, 2013, Nuno K et al., Blood, 2020), indicating that epigenetic changes can have a substantial contribution to acquired drug resistance and refractory disease at relapse. In order to uncover such functional alterations, longitudinal AML patient samples collected at diagnosis, during remission and relapse were analysed with single cell transcriptomics (scRANseq) in order to identify how the molecular wiring of AML cells evolve during disease progression and what alterations make them drug resistant.Methods: scRNAseq was carried out (10x Genomics) on mononuclear cell fractions from bone marrow aspirates of 7 AML patients (from the Finnish Hematology Registry and Clinical Biobank) who received AraC-based induction therapy, achieved CR but later relapsed. Raw data was QC processed (CellRanger), batch corrected (scArches, Harmony). Cell types were identified as described by Granja et al., Nat. Biotechn., 2020 (Fig 1A), the myeloid cell lineage was selected, clusters identified (Leiden) and differentially expressed genes identified (EdgeR). For mechanistic studies primary AML blasts were cultured with bone marrow stromal cells (BMSC) in a system able to recapitulate in vivo chemotherapy resistance (Dhami et al., Br. J, Haematol, 2020).Results: The scRNAseq analysis generated transcriptome for over 67,000 myeloid-lineage cells (hematopoietic/leukemic stem cells (HSC/LSC), multipotent progenitors (MPP), granulocyte-monocyte progenitors (GMP), monocytes (CD14, CD16 subsets), conventional and plasmacytoid dendritic cells; c/pDCs) including 21K, 26K and 22K cells at diagnosis, remission and relapse, respectively (Fig 1B). To identify drug resistant AML clones, we integrated the myeloid lineage cells from the 7 patients and identified clusters (Fig 1C). Of the 24 clusters found, the ones rich in differentiated cells became abundant at remission, while clusters rich in low-differentiation status cells (HSC/LSC, MPP, GMP) diminished. Upon relapse however this trend reversed and all main clusters present at diagnosis returned, indicating that cells from all clusters survived chemotherapy or drug-resistant LSCs could reinstate the full disease spectrum (Fig 1D, 1E). Analysis of AML cell clusters in each patient individually confirmed this pattern with nearly all clusters returning at relapse, excluding the possibility that integration masked patient-level heterogeneity/patient-specific cluster patterns (Fig 1F).Despite the identical cluster distribution between diagnosis and relapse, all 7 patients showed refractory disease at relapse, indicating that small scale molecular alterations, which are not substantial enough to segregate the cells into a new cluster are sufficient to drive drug resistance. To identify these changes, genes differentially expressed and associated gene ontology (GO) terms between diagnosis and relapse were identified for each cluster. The analysis found GO terms commonly regulated in multiple clusters, most notably myeloid cell activation, mitochondrial ATP metabolism, cellular respiration, mRNA maturation and translation.In order to determine whether targeting the protein synthesis machinery, as a process linked to integrated stress response and HSC maintenance, can be exploited to eliminate drug resistant AML cells, we exposed BMSCs to transient proteostatic stress induced by the NEDDylation inhibitor pevonedistat (1 mM for 24 h), cultured primary AML blasts on the stress-conditioned BMSCs and exposed them to drug treatments (Fig 1G). Stress-conditioned BMSCs lost their ability to support AML blast survival and to protect them from tyrosine kinase inhibitors (quizartinib) and the combination of AraC+daunorubicin.Conclusions: We found that AML cell clusters that already exist at diagnosis re-emerge at relapse, although with re-occurring molecular alterations that can provide drug resistance resulting in refractory disease. Targeting one of these pathways, proteostasis, could break the protective interaction between BMSCs and AML blasts resulting in reduced AML survival and enhanced drug sensitivity, representing a potential actionable vulnerability of drug resistant AML cells. [Display omitted] DisclosuresSzegezdi: Bristol Myers Squibb: Research Funding; ONK Therapeutics: Research Funding.

Effect of the Neddylation Inhibitor Pevonedistat on Normal Hematopoietic Stem Cell Subsets and Immune Cell Composition

Introduction: Antitumor activity of the neddylation inhibitor pevonedistat has been documented in several hematologic and non-hematologic malignancies. Unexpectedly, Zhou et al (PNAS, 2016) discovered a dose-dependent biphasic effect of pevonedistat in solid tumor cell lines. While micromolar concentrations inhibited tumor cell growth, low nanomolar concentrations significantly increased cell proliferation and tumor stem cell self-renewal both in vitro and in vivo. The effect of low-dose pevonedistat has not yet been explored in the field of hematopoietic stem cell transplantation. Therefore, we evaluated how pevonedistat affects the viabiilty, growth and proportions of CD34 + cell subpopulations. In view of the emerging role of neddylation in the regulation of both innate and adaptive immunity, we also investigated the influence of pevonedistat on T-cell activation to explore a potentially beneficial effect on posttransplant immune complications.Methods and Results: Using the WST-1 assay we confirmed the biphasic effect of pevonedistat on normal mobilized CD34 + cells. Incubation for 72 h with 0.1 µM pevonedistat significantly increased metabolic activity as a surrogate parameter for proliferation, while 1.0 µM pevonedistat showed a cytotoxic effect. We explored the underlying mechanism for the low-dose effect. Since Zhou et al. previously showed that pevonedistat can promote tumor stem cell proliferation by inducing EGFR homodimerization, we used a proximity ligation assay and found that 0.1 µM pevonedistat induced EGFR homodimerization in normal mobilized CD34 + cells, too. In addition to homodimerization, we also looked at phosphorylation at Tyr1068, a marker of EGFR activation. By flow cytometry, we showed that phosphorylation was increased by 0.01 µM and 0.1 µM pevonedistat. Using an ELISA-based transcription assay, we also observed a biphasic effect of pevonedistat on c-Myc expression, which is regarded as a marker of ‘stemness’. Incubation with pevonedistat for 72 hrs at 0.01 and 0.1 µM stimulated expression of c-Myc, whereas incubation at 1.0 µM downregulated c-Myc.Fractions of hematopoietic stem and progenitor cell (HSPC) subpopulations were measured in CD34 + cells from cord blood after incubation with 0.01, 0.1 and 1.0 µM pevonedistat. Flow cytometry was performed using antibodies against CD34, CD45RA and CD133, as well as 7-AAD for testing cell viability. Exposure to pevonedistat for 72 hrs at 0.1 µM caused an increase in the number of CD34 + cells compared to vehicle-treated CD34+ cells at 72 h as well as compared to initial number of CD34+ cells, whereas 1.0 µM caused a significant decrease. The absolute number of multipotent progenitors (MPP) (CD34 +CD133 +CD45RA -) remained relatively stable at all concentrations, while lympho-myeloid progenitors (LMPP) (CD34+CD133+CD45RA+) and late progenitors (LP) (CD34+CD133-CD45RA+) increased slightly with 0.1 µM pevonedistat compared with controls. However, a significant decrease in LMPP and LP cell numbers was observed at 1.0 µM.Different concentrations of pevonedistat were tested for their capability to modulate allogeneically stimulated T cell activation in a multi-donor mixed lymphocyte reaction (mdMLR) assay in vitro. Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (MSC-EV) were used as internal immuno-modulatory and non-immuno-modulatory controls in the assay. After 5 days, alterations in the immune cell composition were analyzed by flow cytometry. Pevonedistat was not toxic for MNCs in the mdMLR. However, it decreased the number of activated (CD25high CD54+) CD4+ cells and CD8+ cells.Conclusions: One of the problems in the post-transplant period is a rapid decline in MPP numbers, associated with increased risk of engraftment failure. We showed that low-dose pevonedistat (0.1 µM) is capable of increasing the number of CD34 + cells in vitro while keeping the absolute number of MPPs stable. This finding, together with the observed increase in c-Myc expression, suggests that pevonedistat may help to preserve ‘stemness’ of CD34+ donor cells, thus supporting engraftment of hematopoietic stem and progenitor cells. Furthermore, the immunosuppressive effects revealed by mdMLR suggest that low-dose pevonedistat may also play a useful immunomodulatory role in the post-transplant setting to potentially reduce the risk of graft-versus-host disease. [Display omitted] DisclosuresMajidi: Takeda: Research Funding. Germing: Jazz Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Celgene: Honoraria; Novartis: Honoraria, Research Funding; Janssen: Honoraria. Zeiser: Incyte, Mallinckrodt, Novartis: Honoraria, Speakers Bureau. Gattermann: Celgene: Honoraria; Takeda: Research Funding; Novartis: Honoraria.