Chromatin Regulates Genome Targeting with Cisplatin

Article abstract of DOI:10.1002/anie.201701144

Cisplatin derivatives can form various types of DNA lesions (DNA-Pt) and trigger pleiotropic DNA damage responses. Here, we report a strategy to visualize DNA-Pt with high resolution, taking advantage of a novel azide-containing derivative of cisplatin we named APPA, a cellular pre-extraction protocol and the labeling of DNA-Pt by means of click chemistry in cells. Our investigation revealed that pretreating cells with the histone deacetylase (HDAC) inhibitor SAHA led to detectable clusters of DNA-Pt that colocalized with the ubiquitin ligase RAD18 and the replication protein PCNA. Consistent with activation of translesion synthesis (TLS) under these conditions, SAHA and cisplatin cotreatment promoted focal accumulation of the low-fidelity polymerase Polη that also colocalized with PCNA. Remarkably, these cotreatments synergistically triggered mono-ubiquitination of PCNA and apoptosis in a RAD18-dependent manner. Our data provide evidence for a role of chromatin in regulating genome targeting with cisplatin derivatives and associated cellular responses.

Full text of DOI:10.1002/anie.201701144

Angewandte
Communications
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International Edition: DOI: 10.1002/anie.201701144
German Edition: DOI: 10.1002/ange.201701144
Cancer
Chromatin Regulates Genome Targeting with Cisplatin
Emmanouil Zacharioudakis+, Poonam Agarwal+, Alexandra Bartoli, Nathan Abell,
Lavaniya Kunalingam, Valꢀrie Bergoglio, Blerta Xhemalce, Kyle M. Miller,* and
Raphaꢁl Rodriguez*
Dedicated to Professor Stuart L. Schreiber
Abstract: Cisplatin derivatives can form various types of DNA
lesions (DNA-Pt) and trigger pleiotropic DNA damage
responses. Here, we report a strategy to visualize DNA-Pt
with high resolution, taking advantage of a novel azide-
containing derivative of cisplatin we named APPA, a cellular
pre-extraction protocol and the labeling of DNA-Pt by means
of click chemistry in cells. Our investigation revealed that
pretreating cells with the histone deacetylase (HDAC) inhibitor
SAHA led to detectable clusters of DNA-Pt that colocalized
with the ubiquitin ligase RAD18 and the replication protein
PCNA. Consistent with activation of translesion synthesis
(TLS) under these conditions, SAHA and cisplatin cotreatment
promoted focal accumulation of the low-fidelity polymerase
Polh that also colocalized with PCNA. Remarkably, these
cotreatments synergistically triggered mono-ubiquitination of
PCNA and apoptosis in a RAD18-dependent manner. Our
data provide evidence for a role of chromatin in regulating
genome targeting with cisplatin derivatives and associated
cellular responses.
some of the most commonly employed drugs in the clinical
management of cancer.[3] These drugs form covalent bonds
with purine residues to produce toxic DNA-Pt including
mono-adducts, inter- and intra-strand crosslinks. Thus, cellu-
lar responses to cisplatin are pleiotropic and inherently
complex. For example, diverse repair mechanisms including
nucleotide excision repair (NER), base excision repair (BER)
and DNA crosslink repair involving the Fanconi anemia
pathway can process DNA-Pt, and these lesions can also alter
the structure of chromatin or be influenced by chromatin
features.[4] Alternatively, low-fidelity DNA polymerases (e.g.
Polh) can bypass DNA-Pt through a mechanism known as
translesion synthesis, enabling continued replication in the
presence of DNA-Pt, conferring resistance to platinum (Pt)
drugs.[5]
To study DNA-Pt in the context of chromatin, we sought
to develop a surrogate probe that would allow for the
chemical labeling of DNA-Pt in cells. Visual detection of
DNA-Pt with high resolution at the single-cell level can
potentially provide the means to monitor proteins at sites of
lesions and to identify small molecules with a propensity to
modulate targeting with cisplatin derivatives in an unbiased
manner. Prior expertise in characterizing mechanisms of
action of small molecules prompted us to develop an azide-
containing derivative to label DNA-Pt by means of click
chemistry.[1,6] Previous efforts towards the synthesis of
clickable cisplatin derivatives suitable for cell imaging led to
the development of an iminoacridine-Pt complex and a flex-
ible alkyl-azide-containing derivative.[7]
E
merging evidence indicates that functional interactions
between small molecules and genomic DNA are influenced
by chromatin features.[1,2] Cisplatin derivatives represent
[*] Dr. E. Zacharioudakis,[+] Dr. A. Bartoli, L. Kunalingam,
Dr. R. Rodriguez
Institut Curie, PSL Research University
Chemical Cell Biology Group
26 Rue d’Ulm, 75248 Paris Cedex 05 (France)
and
With this in mind, we synthesized the cyclic azidoplati-
num-containing derivative we named 2-aminomethylpyridine
(dichloro) PtII azide (APPA, Figure 1A and Supporting
Information).[8] The design of APPA was inspired from the
structures of cisplatin and picoplatin (Figure 1A), taking
advantage of the aromatic methyl substituent to form a rigid
five-membered ring with Pt. Thus, APPA exhibits a structure
that can form bulky DNA lesions, where the ring prevents
free rotation of the pyridine core chelated to Pt. This
structural distinction was considered important given that
the chemical nature of ligands bound to Pt at sites of lesions in
cells can potentially affect how these lesions are detected and
dealt with.[9] Like cisplatin and picoplatin, APPA exhibited
anti-proliferative properties against human osteosarcoma
U2OS cells (Figure 1B). Thus, we evaluated the reactivity
of APPA towards DNA and our ability to label DNA-Pt with
strained or terminal alkyne-containing fluorophores. A 26-
mer hairpin-forming DNA oligonucleotide containing
CNRS UMR3666, 75005 Paris (France)
and
INSERM U1143, 75005 Paris (France)
and
Institut de Chimie des Substances Naturelles, UPR2301
1 Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex (France)
E-mail: raphael.rodriguez@curie.fr
P. Agarwal,[+] N. Abell, Dr. B. Xhemalce, Dr. K. M. Miller
Department of Molecular Biosciences, Institute of Cellular and
Molecular Biology, University of Texas at Austin
2506 Speedway Stop A5000, Austin, TX 78712 (USA)
E-mail: kyle.miller@austin.utexas.edu
Dr. V. Bergoglio
CRCT, University of Toulouse, INSERM, CNRS, UPS
Avenue Hubert Curien, 31037 Toulouse (France)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201701144.
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Figure 2. A) Detection of labeled DNA-Pt in U2OS cells. B) Schematic
representation of a strategy for enhancing the detection of DNA-Pt in
cells. C) Detection of labeled DNA-Pt in U2OS cells subjected to pre-
extraction. Cells were treated with APPA (250 mm for 3 h). Zoomed
images are 3ꢀ. Scale bar, 20 mm. Unt., untreated.
reflected the targeting of nuclear DNA as well as proteins
and RNA. As a means to selectively detect DNA-Pt, we
implemented a pre-extraction protocol to remove the staining
linked to soluble proteins and RNA targets of Pt drugs
(Figure 2B). This protocol allowed for the detection of
labeled DNA-Pt in the nucleus with some sub-nuclear regions
displaying increased fluorescence intensity (Figure 2C). For
instance, labeled DNA-Pt colocalized with fibrillarin (see
Supporting Information), in agreement with the idea that Pt
drugs target rRNA in the nucleolus.[7b] Collectively, these data
validated APPA as a functional clickable probe suitable for
studying genome targeting with cisplatin derivatives in cells.
Next, we searched for small molecule modulators of
genomic DNA targeting with cisplatin derivatives, using
APPA staining as a readout. We screened a defined set of
small molecules that either operate at the level of chromatin
or that are used in cancer treatment in conjunction with Pt
drugs (Figure 3A and Supporting Information). U2OS cells
were cotreated independently with each small molecule and
APPA, then subjected to chemical labeling and labeled DNA-
Pt were analyzed by fluorescence microscopy. While most
small molecules had no discernable effect on the localization
of DNA-Pt, pre-treatment with the clinically approved DNA
methyl transferase inhibitor azacytidine (5-Aza)[12] and the
histone deacetylase (HDAC) inhibitor SAHA[13] led to the
occurrence of foci of DNA-Pt, indicating the presence of
targeted clusters of purine residues at these sites that can
include mono-adducts, 1,2-GG and 1,3-GTG intra-strand as
well as inter-strand lesions (Figure 3B and C). Western
blotting analysis confirmed that SAHA induced hyperacety-
lation of histone H4 (see Supporting Information), an
established marker of open chromatin.[14] Furthermore, the
structurally distinct Class I HDAC inhibitor MS-275[15] also
led to increased number of DNA-Pt foci, implicating further
the role of HDACs and chromatin in genome targeting with
APPA (see Supporting Information). Importantly, the inten-
sity of DNA-Pt foci was reduced when cells were cotreated
with a two-fold excess of cisplatin acting as a competitor,
indicating that both APPA and cisplatin target similar
Figure 1. A) Molecular structures of APPA, cisplatin and picoplatin.
B) Anti-proliferative activity of cisplatin derivatives against U2OS cells.
C) Schematic representation of a DNA hairpin (hp), a 1,2-GG intra-
strand DNA crosslink (hp-Pt) and chemical labeling of DNA-Pt (hp-Pt-
488). For clarity, a single regioisomer is shown for hp-Pt-488. D) Mass
spectrometry detection of a free DNA hairpin, the corresponding DNA-
Pt and its labeled counterpart. hp-Pt was observed as the molecular
ion peak with loss of nitrogen. E) Molecular structure of APP and
detection of genomic DNA platination by dot blot. DNA samples were
purified from U2OS cells treated with APP (250 mm) or APPA (250 mm)
for 3 h. EtBr (Ethidium bromide) stained gel of input DNA used as
loading control.
a single 1,2-GG dinucleotide, prone to form an intra-strand
crosslink upon treatment with Pt drugs, was incubated with
APPA, purified and then incubated with a strained alkyne-
containing Alexa 488 (Figure 1C).[10] The reaction products
were then analyzed and characterized by mass spectrometry.
We identified three ion peaks corresponding to the free
unreacted hairpin along with the unlabeled and fluorescently
labeled Pt adducts (Figure 1D). Next, we performed similar
experiments in cells using APPA and the control compound 2-
aminomethylpyridine (dichloro) PtII (APP, Figure 1E),
a structurally related active analogue of APPA devoid of
azide functionality and therefore not amenable to labeling.
Labeled genomic DNA-Pt obtained from APPA-treated cells
displayed increased fluorescence compared to equal amounts
of DNA collected from APP-treated cells as monitored by dot
blot (Figure 1E). This data demonstrated that APPA can
form DNA-Pt with genomic DNA in living cells.
With this methodology in hand, we investigated the
localization of DNA-Pt in cells. To this end, cells were treated
with APPA and fixed with formaldehyde prior to labeling
DNA-Pt using copper catalysis. Consistent with previous
observations,[7,11] labeled DNA-Pt exhibited a diffuse cyto-
plasmic and nuclear staining (Figure 2A), which likely
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APPA. Our analysis identified a subset of
genes that were up- or down-regulated by
APPA compared to untreated cells (Fig-
ure 3E and Supporting Information).
Interestingly, the transcriptional changes
measured in cells cotreated with SAHA
and APPA could be attributed to that
observed either in SAHA or APPA inde-
pendent treatments. This supported the
idea that the DNA-Pt clusters detected in
cotreated cells did not trigger additional
transcriptional effects that were unique to
this cotreatment. Furthermore, qPCR
experiments validated that cisplatin
induced similar changes in gene expres-
sion compared to APPA, providing further
evidence that APPA and cisplatin shared
genomic targets (see the Supporting Infor-
mation).
Then, we developed a protocol to
isolate DNA targets of APPA from cells
(Figure 3F). Cells were treated with
APPA Æ SAHA prior to being subjected
to affinity pull-down as previously per-
formed for other classes of molecules.[2,17]
The amount of DNA pulled down from
cells was similar in both experimental
conditions (Figure 3G). This result was
in line with the idea that SAHA did not
solely act by increasing the number of
DNA targets per se, but rather induced
a redistribution of DNA-Pt throughout
the genome, potentiating DNA targeting
at particular sites. This was consistent with
the idea that chromatin relaxation induced
by SAHA altered accessibility of the
genome to APPA, providing additional
insights into how HDAC inhibitors can
sensitize cells to genotoxic drugs.[18]
The occurrence of DNA-Pt foci in
SAHA-treated cells prompted us to deter-
mine whether clusters of lesions could act
as physical roadblocks, potentially affect-
ing DNA-templated processes. Strikingly,
DNA-Pt colocalized with RAD18, an E3
ubiquitin ligase that mediates mono-ubiq-
uitination of the proliferating cell nuclear
antigen PCNA in cells cotreated with
Figure 3. A) Small molecules screened for effects on genome targeting with APPA. B) Fluo-
rescence microscopy detection of labeled DNA-Pt. Arrows indicate DNA-Pt foci. Scale bars,
20 mm. C) Quantification of B. >100 cells scored per condition; n=3; Foci were counted by
means of visual inspection. D) Cisplatin competes with APPA for genome targeting. Zoomed
images are 3ꢀ. Scale bars, 20 mm. E) Heatmap of differentially expressed genes in SAHA
and APPA cotreated cells. Genes were selected based on differential expression in SAHA and
APPA cotreatment compared to untreated. Data from two independent experiments per
condition are shown. Gene expression was monitored by RNA-Seq as described in
Supporting Information. F) Scheme of DNA-Pt pull-down methodology. G) Quantification of
DNA isolated by pull-down from samples treated as indicated; n=3. All experiments
performed in U2OS cells. Cells were treated with APPA (250 mm for 3 h), APP (250 mm for
3 h), SAHA (2.5 mm for 5 h) and cisplatin (500 mm for 5 h). Error bars represent mean ÆSD;
***P<0.001, Student’s t-test; NS, not significant.
genomic loci (Figure 3D). These data suggested that chro-
matin relaxation resulting from SAHA treatment revealed de
novo DNA targets of APPA. Interestingly, DNA-Pt occurring
in SAHA-treated cells did not predominantly colocalize with
the centromere and telomere markers CENP-A and TRF1,
respectively, excluding these loci-containing repetitive
sequences rich in 1,2-purine residues as primary targets of
APPA under these conditions (see Supporting Information).
Given that DNA-Pt can alter transcription,[16] we per-
formed RNA-Seq analyses to evaluate whether SAHA
treatment affected the transcriptional response induced by
SAHA and APPA (Figure 4A). Moreover, clusters of
DNA-Pt also colocalized with PCNA (see Supporting Infor-
mation). Cotreatment with SAHA and APPA led to mono-
ubiquitination of PCNA (Figure 4B), a mark that can
promote the recruitment of low-fidelity polymerases involved
in TLS,[19] and cells cotreated with SAHA and cisplatin or
APPA exhibited increased number of RAD18 foci (see
Supporting Information). Furthermore, treatment with
SAHA and cisplatin led to the focal accumulation of the
TLS polymerase Polh[20] that colocalized with PCNA (Fig-
ure 4C and Supporting Information). Altogether, these data
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tion). These results, along with the RAD18-dependent PCNA
mono-ubiquitination implicated RAD18 in promoting apop-
tosis signaling in response to SAHA and APPA cotreatment.
Comparable results were observed in SAHA and cisplatin
cotreated WT and RAD18 KO cells, demonstrating that this
response was a common feature of these cisplatin derivatives
(Figure 4E). Although the induction of apoptosis signaling by
RAD18 could be potentially counterintuitive owing to the
well-established role of this factor in DNA repair processes
and TLS,[5] HDAC inhibitors have been shown to re-sensitize
cancer cells refractory to cisplatin.[18] Therefore, our results
suggested that the higher level of apoptosis signaling
observed in RAD18 expressing cells in response to SAHA
and cisplatin derivatives is due to the inability of polymerases
to efficiently bypass DNA-Pt clusters. For instance, it is
possible that chromatin relaxation mediated by SAHA
favored the formation of 1,3-GTG intra-strand lesions,
which have previously been shown to block TLS poly-
merases.[21] It is also conceivable that HDAC inhibition
promoted lesion bypass at the expense of DNA repair,[22]
thereby leading to detectable clusters of DNA-Pt prone to
trigger apoptosis through other mechanisms.
Engagement of the replication machinery with DNA-Pt
can promote DNA breaks and cell death. However, cells can
employ DNA damage tolerance pathways involving mono-
ubiquitinated PCNA and specialized low-fidelity polymerases
to bypass DNA-Pt.[5,23] These processes play a critical role in
resistance to Pt drugs.[24] To overcome these mechanisms,
cisplatin derivatives containing bulkier ligands, or combina-
tion therapies with other drugs have been identified.[25] Here,
we have developed a versatile strategy to visualize DNA-Pt in
cells with high resolution. This methodology has allowed the
unbiased identification of small molecule modulators of
genome targeting with cisplatin derivatives. In particular, we
have discovered that treating cells with the HDAC inhibitor
SAHA and APPA resulted in detectable clusters of DNA-Pt
and activation of a DNA damage response at these sites.
While we cannot rule out a putative role of template switching
and homologous recombination that could also be mediated
by RAD18, the increase of Polh foci in cells cotreated with
SAHA and cisplatin is consistent with activation of TLS.
Thus, the response observed in these conditions comes in
agreement with reports showing that clustered DNA lesions
can impair DNA damage response pathways.[26] This study has
uncovered unanticipated insights into how chromatin alter-
ations can affect the targeting of genomic DNA and
sensitization of cancer cells to cisplatin derivatives, establish-
ing a robust experimental platform for basic and translational
research relying on chromatin-targeting small molecules.[27]
Figure 4. A) Colocalization of labeled DNA-Pt with RAD18 in U2OS
cells. Cells were treated with APPA (250 mm for 3 h) and SAHA (2.5 mm
for 5 h). Zoomed images are 3ꢀ. Scale bar, 20 mm. B) Western blot
analysis of PCNA mono-ubiquitination in U2OS cells. Cells were
treated with APPA (250 mm) and SAHA (2.5 mm) as indicated. C) Focal
accumulation of Polh colocalizing with PCNA in U2OS cells. Cells were
treated with cisplatin (10 mm for 3 h) and SAHA (2.5 mm for 5 h).
Zoomed images are 6ꢀ. Scale bar, 20 mm. D) and E) Western blot
analysis of apoptotic markers in WT and RAD18 KO HCT-116 cells.
Cells were treated with APPA (250 mm), SAHA (2.5 mm) and cisplatin
(10 mm) as indicated. FL, full length.
were consistent with the notion that SAHA and cisplatin
derivatives synergistically activated TLS. It is noteworthy that
levels of phosphorylated replication protein A (p-RPA) and
CHK1 (p-CHK1) were similar in APPA-treated and
cotreated cells indicating the absence of additional replication
stress (see Supporting Information). This was in agreement
with the notion that HDAC inhibitors promoted a redistrib-
ution of DNA-Pt in contrast to a global increase in number of
lesions. Remarkably, cotreatment with SAHA and APPA
triggered apoptosis signaling as defined by the cleavage of
PARP and the activation of caspase 3, in several cancer cell
lines including colon carcinoma HCT-116, osteosarcoma
U2OS and ovarian carcinoma A2780 cells (Figure 4D and
Supporting Information). To evaluate whether RAD18 was
directly involved in apoptotic signaling under these condi-
tions, we performed similar experiments with matched HCT-
116 RAD18 knockout (KO) cells. Western blotting indicated
that cells devoid of RAD18 did not display PCNA mono-
ubiquitination in response to SAHA and APPA cotreatment
(Figure 4D). Additionally, markers of apoptosis were not
detected in HCT-116 RAD18 KO cells even though DNA-Pt
foci formed similarly in WT and RAD18 KO cells cotreated
with SAHA and APPA (Figure 4D and Supporting Informa-
Acknowledgements
We thank the PICT-IBiSA@Pasteur Imaging Facility of the
Institut Curie, member of the France-BioImaging national
research infrastructure. Research in the R.R. laboratory is
supported by the European Research Council and the
Emergence Ville de Paris Program. Results incorporated in
this article have received funding from the European
4
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Horizon 2020 research and innovation programme (grant
agreement No [647973]). K.M.M. laboratory is supported by
start-up funds from UT Austin and Cancer Prevention
Research Institute of Texas (CPRIT, grant no. R1116) and
the NIH National Cancer Institute grants RO1 CA198279 and
CA201268. K.M.M. is a CPRIT scholar. The B.X. laboratory
is funded by UT Austin start-up funds and the Welch
Foundation (grant no. F1859). This study also made use of
the Science Park NGS Facility, supported by the CPRIT Core
Facility Support Grant RP120348.
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Conflict of interest
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Keywords: cancer · chromatin · cisplatin · click chemistry ·
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Manuscript received: February 1, 2017
Revised: March 14, 2017
Final Article published: && &&, &&&&
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Cancer
Treatment of cancer cells with cisplatin
derivatives and a histone deacetylase
inhibitor leads to the production of
clusters of platinated DNA lesions that
synergistically activate translesion syn-
thesis and apoptosis signaling. These
findings provide evidence for a role of
chromatin in regulating genome target-
ing with cisplatin derivatives and associ-
ated cellular responses.
E. Zacharioudakis, P. Agarwal, A. Bartoli,
N. Abell, L. Kunalingam, V. Bergoglio,
B. Xhemalce, K. M. Miller,*
R. Rodriguez*
&&&& — &&&&
Chromatin Regulates Genome Targeting
with Cisplatin
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!