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Chang et al. J Cancer Metastasis Treat 2019;5:78 I http://dx.doi.org/10.20517/2394-4722.2019.31 Page 5 of 11

Although hFACT induces extensive, reversible nucleosome unfolding in the presence of either Nhp6
protein or curaxins [Figure 1], the properties of these FACT-unfolded nucleosomes are different [2,31] .
The hFACT-yNhp6-nucleosome forms a reversible complex that is stable during electrophoresis, while
the hFACT-nucleosome complex formed in the presence of curaxins is stable in solution, but falls apart
during PAGE [2,31] . Since Nhp6 and curaxins are expected to bind to similar, end-proximal regions of the
nucleosomal DNA and produce similar unfolding of nucleosomes together with FACT [2,31] , the differences
in stability of the complexes may be explained by the different structures/stabilities of Nhp6-nucleosome
and curaxin-nucleosome complexes, or possibly by a direct effect of Nhp6 protein on the properties of
hFACT.

Many DNA binding molecules other than curaxins also cause trapping of FACT on chromatin in cell-
[40]
free systems . However, to be potent in chromatin destabilization, small molecules need to be able to
get to the nuclei and compete with histones and other chromatin components for DNA binding. The
curaxins that were selected and optimized in a cell-based assay for p53 activation [84,85] easily enter into cell
nuclei and are strong DNA binders. They were also selected in cells which were not responsive to DNA
damage after p53 activation; thus, compounds capable of causing DNA damage were eliminated from the
analysis. Curaxins can cause DNA damage in some systems; e.g., they cause frame shift mutations in some
strains of E. coli in the Ames test. However, the much higher proofreading efficiency of mammalian DNA
polymerases probably mitigates this effect at low concentrations of curaxins and blocks replication at high
[34]
concentrations of the drug. Curaxins are also potent inhibitors of topoisomerases , but, in contrast to
many known drugs that inhibit resolution of topoisomerase cleavage complexes, do not prevent cleavage
itself: curaxins inhibit initiation of topoisomerase action, preventing the induction of DNA breaks. Most
importantly, the toxicity of curaxins for cells does not depend on DNA damage, since DNA damage is
[35]
undetectable in many types of tumor cells as they are being killed by curaxin treatment .
In addition to the c-trapping of FACT, curaxins globally affect the three-dimensional organization of
[46]
the genome in living cells . Studies employing nucleosome arrays show that curaxins induce changes
[46]
in the structure of internucleosomal linker DNA and unfolding of the chromatin fiber . Hi-C analysis
demonstrated that addition of curaxins to cancer cells partially disrupted the boundaries of topologically
associated domains (TADs), inhibited spatial interactions within the TADs (over distances less than 600 Kb),
[46]
and enhanced spatial interactions over longer distances . These alterations of the three-dimensional genome
organization strongly affected enhancer-promoter interactions: only 30% of the intradomain chromatin
interactions annotated in control cells were maintained in CBL0137-treated cells. As a result, numerous
[46]
new alternative, long-range interdomain interactions were detected . Furthermore, genomic studies
demonstrated that curaxins caused partial depletion of CCCTC-binding factor (CTCF) from its binding
[86]
[46]
sites . Since CTCF contributes to maintaining the spatial organization of the mammalian genome ,
depletion of CTCF from domain boundaries by curaxins likely contributed to the drastic changes observed
in genome topology. The changes in chromatin structure, in turn, are expected to dramatically change
gene regulation and the pattern of gene expression. Indeed, after CBL0137 treatment, gene expression
profiles were drastically changed. These changes included downregulation of essential genes necessary for
cell survival, including both normal and translocated versions of MYC family genes, which could explain
[46]
the observed drop in cancer cell viability . Thus, curaxins can be classified as epigenetic drugs that
globally affect gene regulation and cause cancer cell death by targeting the three-dimensional organization
of the genome within living cell nuclei.

THE MECHANISM OF CURAXIN ACTION
Taken together, the current studies suggest the following scenario for curaxin action in cancer cells [Figure 2].
In untreated cancer cells, FACT is present mostly in the nucleoplasm; only a small fraction of the complexes
are transiently associated with nucleosomes perturbed by transcribing RNA polymerase.