Inhibition of EZH2 transactivation function sensitizes solid tumors to genotoxic stress

Drugs that block the activity of the methyltransferase EZH2 are in clinical development for the treatment of non-Hodgkin lymphomas harboring EZH2 gain-of-function mutations that enhance its polycomb repressive function. We have previously reported that EZH2 can act as a transcriptional activator in castration-resistant prostate cancer (CRPC). Now we show that EZH2 inhibitors can also block the transactivation activity of EZH2 and inhibit the growth of CRPC cells. Gene expression and epigenomics profiling of cells treated with EZH2 inhibitors demonstrated that in addition to derepressing gene expression, these compounds also robustly down-regulate a set of DNA damage repair (DDR) genes, especially those involved in the base excision repair (BER) pathway. Methylation of the pioneer factor FOXA1 by EZH2 contributes to the activation of these genes, and interaction with the transcriptional coactivator P300 via the transactivation domain on EZH2 directly turns on the transcription. In addition, CRISPR-Cas9–mediated knockout screens in the presence of EZH2 inhibitors identified these BER genes as the determinants that underlie the growth-inhibitory effect of EZH2 inhibitors. Interrogation of public data from diverse types of solid tumors expressing wild-type EZH2 demonstrated that expression of DDR genes is significantly correlated with EZH2 dependency and cellular sensitivity to EZH2 inhibitors. Consistent with these findings, treatment of CRPC cells with EZH2 inhibitors dramatically enhances their sensitivity to genotoxic stress. These studies reveal a previously unappreciated mechanism of action of EZH2 inhibitors and provide a mechanistic basis for potential combination cancer therapies.

Small Molecules Targeting the Disordered Transactivation Domain of the Androgen Receptor Induce the Formation of Collapsed Helical States

Castration-resistant prostate cancer (CRPC) is a lethal condition suffered by ~35% of prostate cancer patients who become resistant to existing FDA-approved drugs. Small molecules that target the intrinsically disordered N-terminal domain of the androgen receptor (AR-NTD) have shown promise in circumventing CPRC drug-resistance. A prodrug of one such compound, EPI-002, entered human trials in 2015 but was discontinued after phase I due to poor potency. The compound EPI-7170 was subsequently found to have improved potency, and a related compound entered human trials in 2020. NMR measurements have localized the strongest effects of these compounds to a transiently helical region of the disordered AR-NTD but no detailed structural or mechanistic rationale exists to explain their affinity to this region or the comparative potency of EPI-7170. Here, we utilize all-atom molecular dynamics simulations to elucidate the binding mechanisms of the small molecules EPI-002 and EPI-7170 to the disordered AR-NTD. We observe that both compounds induce the formation of collapsed helical states in the Tau-5 transactivation domain and that these bound states consist of heterogenous ensembles of interconverting binding modes. We find that EPI-7170 has a higher affinity to Tau-5 than EPI-002 and that the EPI-7170 bound ensemble contains a substantially higher population of collapsed helical states than the bound ensemble of EPI-002. We identify a network of interactions in the EPI-7170 bound ensemble that stabilize collapsed helical conformations. Our results provide atomically detailed binding mechanisms for EPI compounds consistent with NMR experiments that will prove useful for drug discovery for CRPC.

MDM2-Driven Ubiquitination Rapidly Removes p53 from Its Cognate Promoters

MDM2 is the principal antagonist of the tumor suppressor p53. p53 binds to its cognate DNA element within promoters and activates the transcription of adjacent genes. These target genes include MDM2. Upon induction by p53, the MDM2 protein binds and ubiquitinates p53, triggering its proteasomal degradation and providing negative feedback. This raises the question whether MDM2 can also remove p53 from its target promoters, and whether this also involves ubiquitination. In the present paper, we employ the MDM2-targeted small molecule Nutlin-3a (Nutlin) to disrupt the interaction of MDM2 and p53 in three different cancer cell lines: SJSA-1 (osteosarcoma), 93T449 (liposarcoma; both carrying amplified MDM2), and MCF7 (breast adenocarcinoma). Remarkably, removing Nutlin from the culture medium for less than five minutes not only triggered p53 ubiquitination, but also dissociated most p53 from its chromatin binding sites, as revealed by chromatin immunoprecipitation. This also resulted in reduced p53-responsive transcription, and it occurred much earlier than the degradation of p53 by the proteasome, arguing that MDM2 removes p53 from promoters prior to and thus independent of degradation. Accordingly, the short-term pharmacological inhibition of the proteasome did not alter the removal of p53 from promoters by Nutlin washout. However, when the proteasome inhibitor was applied for several hours, depleting non-conjugated ubiquitin prior to eliminating Nutlin, this compromised the removal of DNA-bound p53, as did an E1 ubiquitin ligase inhibitor. This suggests that the ubiquitination of p53 by MDM2 is necessary for its clearance from promoters. Depleting the MDM2 cofactor MDM4 in SJSA cells did not alter the velocity by that p53 was removed from promoters upon Nutlin washout. We conclude that MDM2 antagonizes p53 not only by covering its transactivation domain and by destabilization, but also by the rapid, ubiquitin-dependent termination of p53–chromatin interactions.

The p53 transactivation domain 1-dependent response to acute DNA damage in endothelial cells protects against radiation-induced cardiac injury

Thoracic radiation therapy can cause endothelial injury in the heart, leading to cardiac dysfunction and heart failure. Although it has been demonstrated that the tumor suppressor p53 functions in endothelial cells to prevent the development of radiation-induced myocardial injury, the key mechanism(s) by which p53 regulates the radiosensitivity of cardiac endothelial cells is not completely understood. Here, we utilized genetically engineered mice that express mutations in p53 transactivation domain 1 (TAD1) (p5325,26) or mutations in p53 TAD1 and TAD2 (p5325,26,53,54) specifically in endothelial cells to study the p53 transcriptional program that protects cardiac endothelial cells from ionizing radiation in vivo. p5325,26,53,54 loses the ability to drive transactivation of p53 target genes after irradiation while p5325,26 can induce transcription of a group of non-canonical p53 target genes, but not the majority of classic radiation-induced p53 targets critical for p53-mediated cell cycle arrest and apoptosis. After 12 Gy whole-heart irradiation, we found that both p5325,26 and p5325,26,53,54 sensitized mice to radiation-induced cardiac injury, in contrast to wild-type p53. Histopathological examination suggested that mutation of TAD1 contributes to myocardial necrosis after whole-heart irradiation, while mutation of both TAD1 and TAD2 abolishes the ability of p53 to prevent radiation-induced heart disease. Taken together, our results show that the transcriptional program downstream of p53 TAD1, which activates the acute DNA damage response after irradiation, is necessary to protect cardiac endothelial cells from radiation injury in vivo.

NF-κB modifies the mammalian circadian clock through interaction with the core clock protein BMAL1

In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.

Respiratory Syncytial virus NS1 protein targets the transactivator binding domain of MED25

Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via an interaction with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the C-terminal α3 helix of NS1 and the MED25 ACID domain. More specifically we showed by NMR that the NS1 α3 sequence primarily binds to the MED25 ACID H2 face, which is a transactivation domain (TAD) binding site for transcription regulators such as ATF6α, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 α3 helix could compete with ATF6α TAD binding to MED25. This finding points to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II.

Non-canonical Function of a Hif-1α Splice Variant Contributes to the Sustained Flight of Locusts

The hypoxia inducible factor (Hif) pathway is functionally conserved across metazoans in modulating cellular adaptations to hypoxia. However, the functions of this pathway under aerobic physiological conditions are rarely investigated. Here, we show that Hif-1α2, a locust Hif-1α isoform, does not induce canonical hypoxic responses but functions as a specific regulator of locust flight, which is a completely aerobic physiological process. Two Hif-1α splice variants were identified in locusts, a ubiquitously expressed Hif-1α1 and a muscle-predominantly expressed Hif- 1α2. Hif-1α1 that induces typical hypoxic responses upon hypoxia exposure, remains inactive during flight. By contrast, the expression of Hif-1α2, which lacks C-terminal transactivation domain, is less sensitive to oxygen-tension but induced extensively by flying. Hif-1α2 sustains flight endurance by promoting glucose oxidation while simultaneously maintaining redox homeostasis by upregulating the production of a reactive oxygen species (ROS) quencher, DJ-1. Overall, this study reveals a novel Hif-mediated mechanism underlying prolonged aerobic physiological activity.

OM301, a Synthetic Polypeptide Containing the p53TA (Transactivation) Domain, Impairs Mitochondrial Activity and Survival of Myeloma Cells

INTRODUCTION: Although the treatment of patients with multiple myeloma (MM) has dramatically improved, those with high-risk characteristics, including the deletion or mutation of the master tumor suppressor gene TP53 on chromosome 17, experience limited survival. OM301 is a synthetic polypeptide containing the p53TA (transactivation) domain, which prevents p53 degradation through inhibition of MDM2. Here, we demonstrate that OM301 has strong anti-MM activity in vitro and in vivo. RESULTS: We first assessed the cytotoxic effects of OM301 in MM cell lines with varying TP53 status (TP53 wild type: MM.1S, H929; TP53 mutated/null: L363, RPMI-8226, U266, JJN3, KMS11) and found that OM301 exerts significant cytotoxic effects at a concentration of ~5 µM in all cell lines we tested, while it was minimally toxic to human peripheral blood mononuclear cells. Next, using immunocompromised NSG mice models injected with MM.1S, we determined the in vivo efficacy of OM301 in three different studies. Many potent anticancer agents, particularly of peptide origin, show prominent anti-tumor effects but fail to sustain similar effects when given intraperitoneally because of poor absorption, distribution, metabolism and excretion properties. OM301 at an intraperitoneal dose of 20 mg/kg/body weight twice a day induced significant reduction in tumor size with respect to vehicle control, suggesting the stability of OM301 without any loss of its activity (n=7, p<0.0001). Accordingly, we investigated its effect in a disseminated NSG/MM.1S model and found that it significantly increased survival (p<0.0001) (see Figure). Because OM301 was designed to simulate the p53 interaction domain with MDM2, we first determined its effect on p53-MDM2 crosstalk using a p53-MDM2 co-Immunoprecipitation (co-IP) assay and compared it with effects from Nutlin-3a, a known inhibitor of p53-MDM2. The co-IP data showed that, unlike Nutlin-3a, OM301 does not inhibit the p53-MDM2 interaction. Thus, to confirm our findings, we first overexpressed MDM2 in HeLa cells, and, using MDM2-IP and p53-MDM2 co-IP, found similar observations. Additionally, OM301 also failed to induce endogenous upregulation of genes activated by p53, such as MDM2 and p21, as opposed to results from Nutlin-3a. RNA sequencing data also showed a distinctive OM301signature, as compared to Nutlin-3a in MM cells. While treatment of Nutlin-3a induced expression of p53-activated canonical genes, OM301-treated cells showed alterations in genes involved in inflammatory responses, c-Myc regulated genes, fatty acid metabolism, glucose metabolism, and oxidative phosphorylation, among others. Next, to dissect its underlying mechanism, we dual-tagged OM301 with fluorophores at the 3' and 5' ends to study its localization and its stability in MM cells. Indeed, OM301 was found to be stable and mainly localized in the cytosol. We then modified OM301 by biotinylation of its penetratin end and first verified its cytotoxic effect in different MM cell lines, which was similar to that of native OM301. The biotinylated OM301 was then immunoprecipitated using streptavidin beads. The streptavidin pull-down and subsequent proteomic analysis confirmed that OM301 does not interact with MDM2 but interacts with c-Myc and with proteins localized in mitochondria, including Bcl-2 and Bcl-2 family members such as Bclaf1, Bcl2L13, and Bcl2L1. Pull-down experiments and immunoblot analysis validated Bcl-2/OM301 interactions. To further evaluate the relative binding potentials of OM301, we performed molecular docking studies using the HPEPDOCK server (Yan et al., Nat Protoc. 2020;15:1829). Post-docking, the calculated docking scores for OM301 was -281, suggesting that OM301directly interacts with Bcl-2. Thus, we evaluated the effects of OM301 on mitochondrial function and physiology. Treatment with OM301 decreased mitochondrial membrane potential in different MM cell lines. OM301 also increased mitochondrial superoxide production and induced mitophagy and mitochondrial fission as seen by electron microscopy. CONCLUSION: Here, we report for the first time that OM301, although designed for p53-selective cells, may instead interact with Bcl-2, which in turn induces mitochondrial dysfunction, leading to cell death irrespective of their TP53 status. Our data suggest that OM301 may be a novel and effective therapeutic option for MM. Figure 1 Figure 1. Disclosures Krishnan: REGENERON: Consultancy; MAGENTA: Consultancy; BMS: Consultancy, Current equity holder in publicly-traded company, Speakers Bureau; JANSSEN: Consultancy, Research Funding; City of Hope Cancer Center: Current Employment; SANOFI: Consultancy; GSK: Consultancy; Amgen: Speakers Bureau. Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

An intrinsic temporal order of c-Jun N-terminal phosphorylation regulates its activity by orchestrating co-factor recruitment

Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-Jun proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-Jun multisite phosphorylation has remained elusive. Here we show that c-Jun phosphorylation by JNK exhibits a defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identified the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis revealed three c-Jun phosphorylation states: unphosphorylated c-Jun recruits the Mbd3 repressor, serine63/73 doubly-phosphorylated c-Jun binds to the Tcf4 co-activator, whereas the fully phosphorylated form disfavours Tcf4 binding attenuating JNK signalling. Thus, c-Jun phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input.