CX-5461 causes nucleolar compaction, alteration of peri- and intranucleolar chromatin arrangement, an increase in both heterochromatin and DNA damage response

Abstract

In this study, we characterize the changes in nucleolar morphology and its dynamics induced by the recently introduced compound CX-5461, an inhibitor of ribosome synthesis. Time-lapse imaging, immunofluorescence and ultrastructural analysis revealed that exposure of cells to CX-5461 has a profound impact on their nucleolar morphology and function: nucleoli acquired a compact, spherical shape and display enlarged, ring-like masses of perinucleolar condensed chromatin. Tunnels consisting of chromatin developed as transient structures running through nucleoli. Nucleolar components involved in rRNA transcription, fibrillar centres and dense fibrillar component with their major constituents ribosomal DNA, RNA polymerase I and fibrillarin maintain their topological arrangement but become reduced in number and move towards the nucleolar periphery. Nucleolar changes are paralleled by an increased amount of the DNA damage response indicator γH2AX and DNA unwinding enzyme topoisomerase I in nucleoli and the perinucleolar area suggesting that CX-5461 induces torsional stress and DNA damage in rDNA. This is corroborated by the irreversibility of the observed altered nucleolar phenotypes. We demonstrate that incubation with CX-5461, apart from leading to specific morphological alterations, increases senescence and decreases cell replication. We discuss that these alterations differ from those observed with other drugs interfering with nucleolar functions.

Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation

“…Our results showed that CX-5461 exhibits the strongest inhibitory effects on c- MYC transcription, which can be attributed to its multifaceted mechanisms of action, including strong c-MYC promoter G4s unfolding inhibition, the ability to intercalate into dsDNA, RNA polymerase I inhibition, chromatin remodeling . Notably, our recent findings indicate that CX-5461 also exhibits a strong affinity for double-stranded DNA ( K d = 0.5 μM) .…”

Nonselective Intercalation of G-Quadruplex-Targeting Ligands into Double-Stranded DNA Quantified by Single-Molecule Stretching

“…For example, CX-5461 is also known as an RNA polymerase inhibitor and induces alternation of heterochromatin arrangement and DNA damage. 66 Further characterization of the G4s' selective stabilization properties is important to the rational design of G4 selective ligands. Notably, PDS and 360A, the two most widely used G4 ligands, showed moderate dsDNA binding (K d = 22.5 and 48.7 μM) and <0.01 ligands/bp intercalation in the presence of 1 μM ligands.…”

“…Even if these ligands have higher affinity to G4s ( K d = 10 –8 –10 –6 M) than dsDNA (Table ), the nonselective binding of G4-targeting ligands to genome dsDNA at micromolar concentrations can be >0.1 ligands/bp and thus may also contribute to cytotoxicity and affect biological processes such as replication and transcription. For example, CX-5461 is also known as an RNA polymerase inhibitor and induces alternation of heterochromatin arrangement and DNA damage . Further characterization of the G4s’ selective stabilization properties is important to the rational design of G4 selective ligands.…”

Targeting the ribosome to treat multiple myeloma

“…1D). In addition to inhibiting Pol I transcription initiation, CX-5461 can also induce alterations in rRNA gene chromatin structure and provoke replication stress that activates ATM/ATR signaling within the nucleoli (18,37,38). Increased phosphorylation of checkpoint kinase 1 (CHK1) at S345 was observed in p53 WT AMO-1 cells while activation of checkpoint kinase 2 (pCHK2 at T68) was absent due to the lack of CHK2 (Fig.…”