We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K⁺. The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed ...parallel-antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5'- and 3'-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K⁺. Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K⁺. The unique human telomeric G-quadruplex structure formed in K⁺ suggests that it can be specifically targeted for anticancer drug design.
Proliferating cell nuclear antigen (PCNA) is an essential component for DNA replication and DNA damage response. Numerous proteins interact with PCNA through their short sequence called the PIP-box ...to be promoted to their respective functions. PCNA supports translesion DNA synthesis (TLS) by interacting with TLS polymerases through PIP-box interaction. Previously, we found a novel small molecule inhibitor of the PCNA/PIP-box interaction, T2AA, which inhibits DNA replication in cells. In this study, we created T2AA analogues and characterized them extensively for TLS inhibition. Compounds that inhibited biochemical PCNA/PIP-box interaction at an IC50 <5μM inhibited cellular DNA replication at 10μM as measured by BrdU incorporation. In cells lacking nucleotide-excision repair activity, PCNA inhibitors inhibited reactivation of a reporter plasmid that was globally damaged by cisplatin, suggesting that the inhibitors blocked the TLS that allows replication of the plasmid. PCNA inhibitors increased γH2AX induction and cell viability reduction mediated by cisplatin. Taken together, these findings suggest that inhibitors of PCNA/PIP-box interaction could chemosensitize cells to cisplatin by inhibiting TLS.
A study conducted from July 2019 to May 2022 at several hospitals in the Western Province, Sri Lanka, focused on dengue virus strains during the COVID-19 pandemic. Among 417 febrile patients, 47% ...were PCR-positive for dengue. Serotyping revealed DENV-1 (12.8%), DENV-2 (46.4%), DENV-3 (37.2%), and DENV-4 (3.6%). Sequencing identified two genotypically distinct variants of DENV-3 and two genotypically distinct variants of DENV-1, while DENV-2 showed a single genotype cluster. Notably, the study found concurrent circulation of two DENV-3 and two DENV-1 genotypes, along with DENV-2, during the pandemic in the area. This data suggests the presence of multiple dengue strains, including several DENV-1 and DENV-3 variants, without major epidemic outbreaks reported in the Western Province. Continuous monitoring and research are essential to understand the dynamics of these dengue strains in the context of the COVID-19 pandemic.
We have previously reported small-molecule inhibitors of Gli1-mediated transcription, an essential down-stream element of the Hh pathway. We created new derivatives of the previous compounds aiming ...to improve the druggable properties. The new compounds, amide conjugates of ketoprofen and indole, showed inhibitory activity and membrane permeability, while also improving the microsome stability. Among them,
33 and
42 inhibited Gli-luciferase reporter in C3H10T1/2 cells that were exogenously transfected with
Gli1 with 2.6
μM and 1.6
μM of IC
50, respectively, and in Rh30 cells that endogenously overexpress
Gli1, and were selective to Gli1 over Gli2.
Formation of the G-quadruplex in the human telomeric sequence can inhibit the activity of telomerase, thus the intramolecular telomeric G-quadruplexes have been considered as an attractive anticancer ...target. Information of intramolecular telomeric G-quadruplex structures formed under physiological conditions is important for structure-based drug design. Here, we report the first structure of the major intramolecular G-quadruplex formed in a native, non-modified human telomeric sequence in K+ solution. This is a hybrid-type mixed parallel/antiparallel-G-stranded G-quadruplex, one end of which is covered by a novel T:A:T triple capping structure. This structure (Hybrid-2) and the previously reported Hybrid-1 structure differ in their loop arrangements, strand orientations and capping structures. The distinct capping structures appear to be crucial for the favored formation of the specific hybrid-type intramolecular telomeric G-quadruplexes, and may provide specific binding sites for drug targeting. Our study also shows that while the hybrid-type G-quadruplexes appear to be the major conformations in K+ solution, human telomeric sequences are always in equilibrium between Hybrid-1 and Hybrid-2 structures, which is largely determined by the 3′-flanking sequence. Furthermore, both hybrid-type G-quadruplexes suggest a straightforward means for multimer formation with effective packing in the human telomeric sequence and provide important implications for drug targeting of G-quadruplexes in human telomeres.
Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most ...human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.
The most common pediatric brain tumors are low-grade gliomas (LGGs). We used whole-genome sequencing to identify multiple new genetic alterations involving BRAF, RAF1, FGFR1, MYB, MYBL1 and genes ...with histone-related functions, including H3F3A and ATRX, in 39 LGGs and low-grade glioneuronal tumors (LGGNTs). Only a single non-silent somatic alteration was detected in 24 of 39 (62%) tumors. Intragenic duplications of the portion of FGFR1 encoding the tyrosine kinase domain (TKD) and rearrangements of MYB were recurrent and mutually exclusive in 53% of grade II diffuse LGGs. Transplantation of Trp53-null neonatal astrocytes expressing FGFR1 with the duplication involving the TKD into the brains of nude mice generated high-grade astrocytomas with short latency and 100% penetrance. FGFR1 with the duplication induced FGFR1 autophosphorylation and upregulation of the MAPK/ERK and PI3K pathways, which could be blocked by specific inhibitors. Focusing on the therapeutically challenging diffuse LGGs, our study of 151 tumors has discovered genetic alterations and potential therapeutic targets across the entire range of pediatric LGGs and LGGNTs.
Low-grade neuroepithelial tumors (LGNTs) are diverse CNS tumors presenting in children and young adults, often with a history of epilepsy. While the genetic profiles of common LGNTs, such as the ...pilocytic astrocytoma and ‘adult-type’ diffuse gliomas, are largely established, those of uncommon LGNTs remain to be defined. In this study, we have used massively parallel sequencing and various targeted molecular genetic approaches to study alterations in 91 LGNTs, mostly from children but including young adult patients. These tumors comprise dysembryoplastic neuroepithelial tumors (DNETs;
n
= 22), diffuse oligodendroglial tumors (d-OTs;
n
= 20), diffuse astrocytomas (DAs;
n
= 17), angiocentric gliomas (
n
= 15), and gangliogliomas (
n
= 17). Most LGNTs (84 %) analyzed by whole-genome sequencing (WGS) were characterized by a single driver genetic alteration. Alterations of
FGFR1
occurred frequently in LGNTs composed of oligodendrocyte-like cells, being present in 82 % of DNETs and 40 % of d-OTs. In contrast, a
MYB
-
QKI
fusion characterized almost all angiocentric gliomas (87 %), and
MYB
fusion genes were the most common genetic alteration in DAs (41 %). A BRAF:p.V600E mutation was present in 35 % of gangliogliomas and 18 % of DAs. Pathogenic alterations in
FGFR1/2/3
,
BRAF
, or
MYB/MYBL1
occurred in 78 % of the series. Adult-type d-OTs with an
IDH1/2
mutation occurred in four adolescents, the youngest aged 15 years at biopsy. Despite a detailed analysis, novel genetic alterations were limited to two fusion genes,
EWSR1
-
PATZ1
and
SLMAP
-
NTRK2
, both in gangliogliomas. Alterations in BRAF, FGFR1, or MYB account for most pathogenic alterations in LGNTs, including pilocytic astrocytomas, and alignment of these genetic alterations and cytologic features across LGNTs has diagnostic implications. Additionally, therapeutic options based upon targeting the effects of these alterations are already in clinical trials.
Of nine ependymoma molecular groups detected by DNA methylation profiling, the posterior fossa type A (PFA) is most prevalent. We used DNA methylation profiling to look for further molecular ...heterogeneity among 675 PFA ependymomas. Two major subgroups, PFA-1 and PFA-2, and nine minor subtypes were discovered. Transcriptome profiling suggested a distinct histogenesis for PFA-1 and PFA-2, but their clinical parameters were similar. In contrast, PFA subtypes differed with respect to age at diagnosis, gender ratio, outcome, and frequencies of genetic alterations. One subtype, PFA-1c, was enriched for 1q gain and had a relatively poor outcome, while patients with PFA-2c ependymomas showed an overall survival at 5 years of > 90%. Unlike other ependymomas, PFA-2c tumors express high levels of OTX2, a potential biomarker for this ependymoma subtype with a good prognosis. We also discovered recurrent mutations among PFA ependymomas. H3 K27M mutations were present in 4.2%, occurring only in PFA-1 tumors, and missense mutations in an uncharacterized gene,
CXorf67
, were found in 9.4% of PFA ependymomas, but not in other groups. We detected high levels of wildtype or mutant
CXorf67
expression in all PFA subtypes except PFA-1f, which is enriched for H3 K27M mutations. PFA ependymomas are characterized by lack of H3 K27 trimethylation (H3 K27-me3), and we tested the hypothesis that CXorf67 binds to PRC2 and can modulate levels of H3 K27-me3. Immunoprecipitation/mass spectrometry detected EZH2, SUZ12, and EED, core components of the PRC2 complex, bound to CXorf67 in the Daoy cell line, which shows high levels of
CXorf67
and no expression of H3 K27-me3. Enforced reduction of CXorf67 in Daoy cells restored H3 K27-me3 levels, while enforced expression of CXorf67 in HEK293T and neural stem cells reduced H3 K27-me3 levels. Our data suggest that heterogeneity among PFA ependymomas could have clinicopathologic utility and that
CXorf67
may have a functional role in these tumors.
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