TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis

Human telomerase is a specialized DNA polymerase whose catalytic core includes both TERT and human telomerase RNA (hTR). Telomerase in humans, which is silent in most somatic cells, is activated to maintain the telomere length (TEL) in various types of cancer cells, including melanoma. In the vast majority of tumor cells, the TERT promoter is mutated to promote proliferation and inhibit apoptosis. Here, we exploited NG-ABEmax to revert TERT -146 T to -146 C in melanoma, and successfully obtained TERT promoter revertant mutant cells. These TERT revertant mutant cells exhibited significant growth inhibition both in vitro and in vivo. Moreover, A375−146C/C cells exhibited telomere shortening and the downregulation of TERT at both the transcription and protein levels, and migration and invasion were inhibited. In addition, TERT promoter revertant mutation abrogated the inhibitory effect of mutant TERT on apoptosis via B-cell lymphoma 2 (Blc-2), ultimately leading to cell death. Collectively, the results of our work demonstrate that reverting mutations in the TERT promoter is a potential therapeutic option for melanoma.

Porphyrin in prebiotic catalysis: Ascertaining a route for the emergence of early metalloporphyrins

Metal ions are known to catalyze certain prebiotic reactions. However, the transition from metal ions to extant metalloenzymes remains unclear. Porphyrins are found ubiquitously in the catalytic core of many ancient metalloenzymes. In this study, we evaluated the influence of porphyrin-based organic scaffold, on the catalysis, emergence and putative molecular evolution of prebiotic metalloporphyrins. We studied the effect of porphyrins on the transition metal ion-mediated oxidation of hydroquinone (HQ). We report a change in the catalytic activity of the metal ions in the presence of porphyrin. This was observed to be facilitated by the coordination between metal ions and porphyrins or by formation of non-coordinated complexes. The metal-porphyrin complexes also oxidized NADH, underscoring its versatility at oxidizing more than one substrate. Our study highlights the selective advantage that some of the metal ions would have had in the presence of porphyrin, underscoring their role in shaping the evolution of protometalloenzymes.

Structural model of PORCN illuminates disease-associated variants and drug binding sites

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Based on homology to mammalian MBOAT proteins we developed and validated a structural model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model enhances our mechanistic understanding of PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and can facilitate the development of improved inhibitors and the understanding of disease relevant PORCN mutants.

Zipper head mechanism of telomere synthesis by human telomerase

Telomerase, a multi-subunit ribonucleoprotein complex, is a unique reverse transcriptase that catalyzes the processive addition of a repeat sequence to extend the telomere end using a short fragment of its own RNA component as the template. Despite recent structural characterizations of human and Tetrahymena telomerase, it is still a mystery how telomerase repeatedly uses its RNA template to synthesize telomeric DNA. Here, we report the cryo-EM structure of human telomerase holoenzyme bound with telomeric DNA at resolutions of 3.5 Å and 3.9 Å for the catalytic core and biogenesis module, respectively. The structure reveals that a leucine residue Leu980 in telomerase reverse transcriptase (TERT) catalytic subunit functions as a zipper head to limit the length of the short primer–template duplex in the active center. Moreover, our structural and computational analyses suggest that TERT and telomerase RNA (hTR) are organized to harbor a preformed active site that can accommodate short primer–template duplex substrates for catalysis. Furthermore, our findings unveil a double-fingers architecture in TERT that ensures nucleotide addition processivity of human telomerase. We propose that the zipper head Leu980 is a structural determinant for the sequence-based pausing signal of DNA synthesis that coincides with the RNA element-based physical template boundary. Functional analyses unveil that the non-glycine zipper head plays an essential role in both telomerase repeat addition processivity and telomere length homeostasis. In addition, we also demonstrate that this zipper head mechanism is conserved in all eukaryotic telomerases. Together, our study provides an integrated model for telomerase-mediated telomere synthesis.

Histone Acetyltransferase (HAT) Activator, YF2, Modulates the p53:BCL6 Axis and Antigen Presentation in Diffuse Large B-Cell Lymphomas

A hallmark of DLBCL is epigenetic derangements characterized by monoallelic mutations in histone acetyltransferases (HATs); EP300 (p300) and CREBBP (CBP). The intact allele offers the opportunity for targeted therapies designed to overcome mutational dysregulation. We reported the discovery of YF2, a first-in-class HAT activator that demonstrates selective cytotoxicity in HAT-mutated DLBCL and induces HAT-mediated histone acetylation in vitro and in vivo. Here, we detail the mechanisms of action and the downstream effects of YF2 treatment. A unique feature of CBP/p300 is that it harbors a regulatory loop within its catalytic domain that undergoes autoacetylation which is critical for maintaining normal function. In order to determine if YF2 is able to induce the autoacetylation of p300/CBP, thereby increasing its catalytic activity, hypoacetylated CBP/p300 was combined with YF2 and Ac-CoA. YF2 demonstrated significant induction of CBP/p300 autoacetylation. To understand how YF2 interacts with HATs we analyzed the thermal stability, via thermal shift assay, of CBP/p300 subunits in the presence of YF2. We observed a T m shift when utilizing the full p300 (ΔT m = -2.9 oC)/CBP(ΔT m = -3.4 oC) catalytic core, which includes the catalytic, PHD/RING, and bromodomain. YF2 does not interact directly with the catalytic domain as there were no observed T m shift. YF2 significantly interacts with the bromodomain (ΔT m = -5.6 oC). In silico analysis has shown that the bromodomain has 3 TRP domains that are predicted to interact with small molecules. Next, we sought to determine how resistance to HDAC inhibitors (HDACi) and mutations/loss of HATs affects sensitivity to YF2. We first developed cell lines to be 10-fold resistant to HDACi romidepsin. When treated with YF2, resistant-SUDHL-6 was more sensitive to YF2 than the parental cell line (Resistant IC 50 = 2.2µM vs Parental IC 50 = 7.22µM). We found no change in YF2 sensitivity in the HAT wt OCI-Ly1 cell line. We performed CRISPR KO of EP300 in wt OCI-Ly7 cell line. A single cell clone with EP300 mutations was identified (OCI-Ly7-EP300 +/-). ICE analysis revealed that the percentage of indels was 12%. OCI-Ly7-EP300 +/- had lower p300 protein expression and were more sensitive to YF2 (IC 50 = 14.05µM) compared to wt (IC 50 = 23.7µM) when measured by Annexin V and CellTiter Glo assay. CBP/p300 is involved in the transcriptional activation of p53 through direct acetylation. YF2 induced both CBP (EC 50 = 15.47µM) and p300 (EC 50 = 6.05µM) mediated acetylation of p53 in cell free assays. As measured by RNA-Seq, YF2 altered multiple pathways regulated by CBP/p300 such as apoptosis and the p53 pathways. The p53 pathway was significantly upregulated in all cell lines. Validation of this pathway via qPCR, revealed p21, BAI1, ATM, FAS, FOS were upregulated in all cell lines. Additionally, YF2 induced G2/M arrest in a dose dependent manner when assessed via flow cytometry. We also observed modest increases in p21 and decrease CCND1 expression with YF2 treatment. BCL6, a transcriptional repressor linked to B-cell lymphomagenesis, is in part regulated through acetylation by CBP/p300. Mechanistically, CBP and the BLC6/SMRT/HDAC3 repressor complex co-occupy enhancers in the MHC Class II loci. Lack of functional CBP drives BCL6 mediated MHC repression resulting in reduced MHC gene expression and altered antigen presentation. In cell free assays, we YF2 induced p300 mediated BCL6 acetylation (IC 50 = 1.58 µM). We hypothesized HAT activation by YF2 could increase MHC expression in DLBCL. RNA-Seq analysis revealed YF2 led to upregulation of the interferon gamma pathway. Significantly, cell lines treated with YF2 showed increased MHC Class I and II expression when analyzed via flow cytometry. In summary, these findings demonstrate that YF2 interacts with the RING and bromodomains, leading to an allosteric change within the catalytic pocket to facilitate increased acetylation. In addition, YF2 leads to CBP/p300 autoacetylation, further enhancing enzymatic activity. We also demonstrated that YF2 is highly selective to DLBCL harboring HAT mutations and overcomes resistance to HDACi. Additionally, YF2 treatment modulates the p53:BCL6 axis, cell cycle progression, and antigen presentation pathway potentially restoring immune surveillance. These results support future clinical application of YF2 in HAT mutated lymphomas. Figure 1 Figure 1. Disclosures Amengual: Seagen: Consultancy; Daiichi Sankyo, Inc: Consultancy; Epizyme, Inc.: Speakers Bureau; Appia Pharmaceuticals: Research Funding.

Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

Secondary-structure switch regulates the substrate binding of a YopJ family acetyltransferase

The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-β-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.

Catalytic core–shell nanoparticles with self-supplied calcium and H2O2 to enable combinational tumor inhibition

Nanoparticles, presenting catalytic activity to induce intracellular oxidative species, have been extensively explored for tumor treatment, but suffer daunting challenges in the limited intracellular H2O2 and thus suppressed therapeutic efficacy. Here in this study, a type of composite nanoparticles, consisting CaO2 core and Co-ferrocene shell, is designed and synthesized for combinational tumor treatment. The findings indicate that CaO2 core can be hydrolyzed to produce large amounts of H2O2 and calcium ions at the acidic tumor sites. Meanwhile, Co-ferrocene shell acts as an excellent Fenton catalyst, inducing considerable ROS generation following its reaction with H2O2. Excessive cellular oxidative stress triggers agitated calcium accumulation in addition to the calcium ions released from the particles. The combined effect of intracellular ROS and calcium overload causes significant tumor inhibition both in vitro and in vivo.

Role of the Topoisomerase IIα Chromatin Tether domain in Nucleosome Binding & Chromosome Segregation

Due to the intrinsic nature of DNA replication, replicated genomes retain catenated genomic loci that must be resolved to ensure faithful segregation of sister chromatids in mitosis. Type II DNA Topoisomerase (TopoII) decatenates the catenated genomic DNA through its unique Strand Passage Reaction (SPR). Loss of SPR activity results in anaphase chromosome bridges and formation of Polo-like Kinase Interacting Checkpoint Helicase (PICH)-coated ultra-fine DNA bridges (UFBs) whose timely resolution is required to prevent micronuclei formation. Vertebrates have two TopoII isoforms– TopoIIα and TopoIIβ, that share a conserved catalytic core. However, the essential mitotic function of TopoIIα cannot be compensated by TopoIIβ, due to differences in their catalytically inert C-terminal domains (CTDs). Using genome-edited human cells, we show that specific binding of TopoIIα to methylated histone, tri-methylated H3K27 (H3K27me3), via its Chromatin Tether (ChT) domain within the CTD contributes critically to avoid anaphase UFB formation. Reducing H3K27 methylation prior to mitosis increases UFBs, revealing a requirement for proper establishment of H3K27me3 after DNA replication to facilitate TopoIIα-ChT dependent UFB prevention. We propose that interaction of the TopoIIα-ChT with H3K27me3 is a key factor that ensures the complete resolution of catenated loci to permit faithful chromosome segregation in human cells.