Nucleolar Stress Induction by Oxaliplatin and Derivatives

Article abstract of DOI:10.1021/jacs.9b10319

Platinum(II) compounds are a critical class of chemotherapeutic agents. Recent studies have highlighted the ability of a subset of Pt(II) compounds, including oxaliplatin but not cisplatin, to induce cytotoxicity via nucleolar stress rather than a canonical DNA damage response. In this study, influential properties of Pt(II) compounds were investigated using redistribution of nucleophosmin (NPM1) as a marker of nucleolar stress. NPM1 assays were coupled to calculated and measured properties such as compound size and hydrophobicity. The oxalate leaving group of oxaliplatin is not required for NPM1 redistribution. Interestingly, although changes in diaminocyclohexane (DACH) ligand ring size and aromaticity can be tolerated, ring orientation appears important for stress induction. The specificity of ligand requirements provides insight into the striking ability of only certain Pt(II) compounds to activate nucleolar processes.

Full text of DOI:10.1021/jacs.9b10319

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Communication
Nucleolar Stress Induction by Oxaliplatin and Derivatives
Emily C. Sutton, Christine E. McDevitt, Jack Y. Prochnau, Matthew V. Yglesias, Austin M. Mroz,
Min Chieh Yang, Rachael M. Cunningham, Christopher H. Hendon, and Victoria J. DeRose
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b10319 • Publication Date (Web): 31 Oct 2019
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Nucleolar Stress Induction by Oxaliplatin and Derivatives
Emily C. Sutton#,†,‡, Christine E. McDevitt#,§, Jack Y. Prochnau§, Matthew V. Yglesias‡§, Austin M.
Mroz§Ⅱ, Min Chieh Yang§Ⅱ, Rachael M. Cunningham§, Christopher H. Hendon§Ⅱ, Victoria J.
DeRose*,‡,§,Ⅱ
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†Department of Biology, ‡Institute of Molecular Biology, §Department of Chemistry
and Biochemistry, Materials Science Institute — University of Oregon, Eugene, OR
97403
Ⅱ
Supporting Information Placeholder
Ribosome biogenesis occurs in the nucleolus, a con-
served and highly structured organelle in eukaryotes.
Disruptions of the nucleolus or ribosome biogenesis
trigger the nucleolar stress response, which leads to
cell death or senescence via activation of the tumor
suppressor p53. Because its molecular mechanisms
are not yet fully understood, and due to its potential
role as a chemotherapeutic target, this fascinating
stress process is an area of intense interest in the
fields of molecular biology and medicine.4–7
ABSTRACT: Platinum(II) compounds are a critical
class of chemotherapeutic agents. Recent studies
have highlighted the ability of a subset of Pt(II) com-
pounds, including oxaliplatin but not cisplatin, to in-
duce cytotoxicity via nucleolar stress rather than a
canonical DNA damage response. In this study, influ-
ential properties of Pt(II) compounds were investi-
gated using redistribution of nucleophosmin (NPM1)
as a marker of nucleolar stress. NPM1 assays were
coupled to calculated and measured properties such
as compound size and hydrophobicity. The oxalate
leaving group of oxaliplatin is not required for NPM1
redistribution. Interestingly, although changes in
diaminocyclohexane (DACH) ligand ring size and
aromaticity can be tolerated, ring orientation ap-
pears important for stress induction. The specificity
of ligand requirements provides insight into the
striking ability of only certain Pt(II) compounds to
activate nucleolar processes.
The specificity of oxaliplatin as a nucleolar stress
inducer is intriguing when considered alongside
other data indicating a relationship between Pt(II)
compounds and the nucleolus.8 Post-treatment fluo-
rescent labeling of clickable Pt(II) drug analogs has
shown localization of these compounds to the nu-
cleolus,8,9 and there is significant evidence that Pt(II)
compounds associate with ribosomes and ribosomal
RNA (rRNA).10–16 The structural determinants and
molecular mechanisms by which only specific Pt(II)
compounds may cause a nucleolar stress response
are not understood. Here, we explore properties of
oxaliplatin and other Pt(II) compounds and find that
a narrow window of derivatives is able to induce nu-
cleolar stress. The results define a set of constraints
for Pt(II) compounds to induce this unique cell death
pathway.
The chemotherapeutic agent cisplatin has inspired
the synthesis and investigation of thousands of Pt(II)
analogs.1 Of these, only two other compounds — car-
boplatin and oxaliplatin — have met FDA standards
for medical use. Until recently, it was believed that
the cytotoxicity of these compounds could be attrib-
uted solely to their DNA crosslinking abilities and
subsequent induction of the DNA Damage Response
(DDR), a known trigger of apoptotic pathways.2 As
the body of research on Pt(II) reagents has grown, a
more complex picture has emerged of the mecha-
nisms of action behind these ubiquitous drugs.2 A
striking recent discovery is that oxaliplatin, but not
cisplatin or carboplatin, causes cytotoxicity via dis-
ruptions in ribosome biogenesis rather than DDR.3
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Cells were treated for 24 hours with a given com-
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pound prior to fixation and secondary immunofluo-
rescence to detect NPM1 (Figure 2 and S1). The ex-
tent of NPM1 redistribution was quantified using an
image analysis pipeline (Figure S1) to calculate the
coefficient of variation (CV) of NPM1 intensity in
each cell (Figure 3). The uniform distribution of
NPM1 in cells undergoing nucleolar stress yields a
low CV, as seen in positive control samples treated
with known stress-inducer Actinomycin D (Figure 3).
In addition to the observation of NPM1 redistribu-
tion, we noted a change in the shape of nucleoli from
eccentrically shaped aggregates to round, sphere-like
structures upon stress induction (Figure 2).
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As predicted,21,22 oxaliplatin (2) induces robust re-
distribution of NPM1, similar to the positive control,
while NPM1 distribution in cisplatin (1) and car-
boplatin (3) treated cells more closely resembles that
of the no-treatment control (Figure 2, Figure 3).
Figure 1. Compounds tested for inducing nucleolar
stress via NPM1 relocalization in mammalian cells.
We selected Pt(II) compounds to test a variety of
properties including steric bulk, hydrophobicity,
crosslinking ability, and ligand orientation (Figure 1).
The extent of nucleolar stress was measured by nu-
cleophosmin (NPM1) imaging (Figure 2 and Figure
S1). Translocation of NPM1 from the granular com-
ponent (GC) of the nucleolus to the nucleoplasm is a
hallmark of the nucleolar stress response.17,18 This
translocation has been shown to be a necessary, but
not sufficient, feature of p53-mediated cell death
upon nucleolar stress,18 and thus is a robust and ap-
propriate marker for nucleolar stress. A549 cells
were selected for this study as they are well estab-
lished to have a characteristic nucleolar stress re-
sponse resulting in p53-mediated apoptosis.19,20
We note that for cisplatin-treated cells, a small
amount of NPM1 redistribution was observed at this
treatment concentration. This is likely because the
24-hour IC-50 value of cisplatin (12.8 µM, Table S1)
is close to the treatment concentration, which may
result in a subset of cisplatin-treated cells experienc-
ing abnormal NPM1 distribution downstream of
other cell death pathways, such as those mediated by
the DDR (see footnote1). Oxaliplatin, by contrast,
shows robust NPM1 relocalization at treatment con-
centrations well below the 24-hr IC-50 value (81.5
µM, Table S1), suggesting that nucleolar stress sig-
nificantly precedes cell death pathways.21 NPM1 re-
localization at concentrations below IC-50 values was
observed with other stress-inducing compounds,
some of which do not exhibit significant cell death
until 48 hours of treatment (Tables S1 and S2). Thus,
observation of nucleolar stress at 24 hours does not
necessarily predict measured toxicity.
1
Footnote: This model is supported by previously
published data demonstrating that cisplatin causes sig-
nificantly more DNA damage than oxaliplatin23–25 and
that DDR-mediated cell death occurs upon cisplatin
treatment, but not oxaliplatin treatment.3 Additionally,
data from our lab shows that DNA damage occurs at
early time points in cisplatin-treated cells, prior to any
putative NPM1 relocation, whereas no such damage
occurs prior to observed NPM1 distribution in ox-
aliplatin-treated cells (unpublished).
Figure 2. Nucleolar stress induced by Pt(II) com-
pounds. NPM1 (green) relocalization following 24-hr
treatment in A549 cells. Treatment concentrations
are 10 µM except for Actinomycin D (5 nM). Scale bar
= 10 µm.
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small molecule did not induce stress in A549 cells
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(Figures 2 and 3), indicating that a chelating diamine
ligand, a common feature between PtEn, DACHPt,
and oxaliplatin, is not sufficient to induce stress. The
addition of a methyl group to generate the bulkier
PtMeEn (8), was also not sufficient to induce stress
(Figure 3, Figure S2, Figure S3). Compound 9, penta-
platin, possesses a five-membered ring that places its
volume between the non-stress inducing PtMeEn and
the stress-inducing six-membered DACHPt. Penta-
platin was found to induce nucleolar stress (Figure
1), although with a slightly higher resultant CV than
positive controls or oxaliplatin (Fig. 3). These results
suggest that bulk may be an important metric lending
towards the ability of Pt(II) compounds to induce
nucleolar stress. Using computed values for volume,
we conclude that as a general trend, Pt(II) com-
pounds with more steric bulk are more likely to in-
duce nucleolar stress (Figure 4a, Y axis). Compound
length, or steric reach, also generally appears to cor-
relate with stress induction (Figure 4a, X axis). Some
exceptions to this trend are discussed below.
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Figure 3. Quantification of NPM1 relocalization induced
by Pt(II) compounds. Treatment conditions as in Figure
2; replicates, CV calculations, and boxplot presentation
as described in SI. For each treatment data set, boxes
represent median, first and third quartiles, and vertical
lines are the range of data with outliers defined in the
SI.
Oxaliplatin is distinct from cisplatin and car-
boplatin in both labile and non-labile Pt(II) ligands.
The labile, chelating oxalate ligand of oxaliplatin de-
lays aquation and therefore biomolecule crosslink-
ing26 in comparison with cisplatin. We exchanged the
labile and non-labile ligands of oxaliplatin and cis-
platin with compounds 4 and 5. We found that com-
pound 4, DACHPt, which has the non-labile DACH
ligand of oxaliplatin and labile chloride groups of cis-
platin, induces robust nucleolar stress. By compari-
son, 5, or DOAP, which possesses the non-labile am-
mine ligands of cisplatin and the labile oxalic acid
ligand of oxaliplatin, does not induce stress (Figures
2 and 3). The oxalic acid ligand alone also had no in-
fluence on NPM1 redistribution, nor did the DACH
ligand by itself (Figure S2, Figure S3). From this, we
concluded that the non-labile DACH ligand of ox-
aliplatin is responsible for the nucleolar stress re-
sponse.
The chair confirmation of the DACH ligand is not
essential for stress induction. BenzaPt (10), in which
the DACH cyclohexane is replaced with a planar aro-
matic ring (Figure 5B), also induces robust NPM1
redistribution (Figure 2 and Figure 3). Like DACHPt,
BenzaPt is more hydrophobic than the simpler
diam(m)ine compounds. To estimate the relative hy-
drophobicity of our compounds of interest, we meas-
ured their water/octanol partition coefficients (Sup-
plementary materials and methods). All of the stress-
inducing compounds were found to be relatively hy-
drophobic (Figure 4b), leading to the conclusion that
hydrophobicity, like steric bulk, positively correlates
with stress induction. Similarly to steric bulk, how-
ever, exceptions to this trend were observed.
Compounds 11, 12, and 13 do not cause NPM1 re-
localization despite being similar or higher in terms
of size and hydrophobicity to compounds that do
cause nucleolar stress (Figure 4). These exceptions
may provide insight into the elements responsible for
causing stress.
We next considered whether crosslinking of bio-
molecules by the Pt(II) compound is necessary for
the induction of nucleolar stress. An alternate hy-
pothesis is that the charged Pt(II) acts as a targeting
agent to facilitate transport of the DACH moiety to
the nucleolus where it disrupts nucleolar processes
without forming a Pt(II)-DACH lesion on a bio-
molecule. Compound 6, DACH-En, retains the DACH
ligand but is unable to form crosslinks with bio-
molecules due to replacement of the oxalic acid with
an ethylenediamine ligand (Figure 1). This positively
charged compound did not induce stress (Figure 2,
Figure 3), suggesting that crosslinking of Pt(II) to
cellular targets is necessary to induce a nucleolar
stress response.
To refine requirements of the non-labile Pt(II)
ligand that cause nucleolar stress, we examined the
effects of steric bulk by testing 7, 8 and 9. Pt-En (7)
possesses a non-labile ethylenediamine ligand. This
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Figure 5. (A) Computed volume and distance meas-
urements for non stress-inducing compounds 11 and
12 alongside stress-inducing compounds 4 and 10. (B)
Ball and stick drawings of non stress-inducing com-
pound 8 alongside stress-inducing 4, 9, and 10.
Figure 4. Size and hydrophobicity correlate with stress
induction, with some exceptions. Compounds with a
higher (less negative) partition coefficient are more
hydrophobic than those with a lower (more negative)
partition coefficient. Measurements and calculations are
described in SI.
Taken together, the results described provide sig-
nificant insight into the structural determinants of
nucleolar stress induction among Pt(II) compounds.
We conclude that there is an important role for
ligand orientation and a general correlation between
steric bulk and stress induction (Figure 5).
One particularly interesting comparison is between
APP (12)27,28 and BenzaPt (10) (Figure 5A). These
two Pt(II) compounds both have an aromatic ring,
but differ in the orientation of the ring relative to the
Pt(II), and by extension ring orientation relative to a
biomolecule to which the compound is bound. While
BenzaPt causes nucleolar stress, APP does not. Simi-
larly, picoplatin (11) does not cause nucleolar stress
despite having volume and reach similar to other
compounds (Figure 4 and Figure 5). These results
demonstrate a critical role for ring orientation in the
ability of Pt(II) compounds to induce nucleolar
stress.
The differential responses induced by these com-
pounds have clinical implications as the three cur-
rently FDA-approved Pt(II) chemotherapeutics are
known to have different treatment and side effect
profiles. Other important differences between these
compounds have been observed in the literature. For
example, oxaliplatin is noted to cause immunogenic
cell death (ICD), while cisplatin does not.29–31 Al-
though this contrast is also observed in nucleolar
stress, connections between ICD and nucleolar stress
are not well-studied. Oxaliplatin has also been shown
to cause changes in the size of neuronal nucleoli cor-
relating with peripheral neuropathy,32 a common
side effect associated with oxaliplatin chemotherapy
regimens. The relationship between nucleolar stress
and platinum-induced neurotoxicity has not been
explored. Additionally, there is some evidence that
p53 mutations in colon cancer cell lines result in re-
sistance to oxaliplatin-mediated cell death.33 This
may be of interest given oxaliplatin’s use in colon
cancer treatments and p53’s role in nucleolar stress-
induced cell death.
The observation that azidoplatin (13) does not
cause stress is of interest as this compound has ex-
tended volume and has previously been shown to
localize to the nucleolus.9 Thus, nucleolar localiza-
tion, even when combined with relatively high hy-
drophobicity and larger bulk and length, is not suffi-
cient to induce nucleolar stress.
Further study is warranted to provide clarification
on the molecular mechanisms by which these com-
pounds induce such different responses in the cell.
For example, the stress-inducers may be interfering
with progression of ribosome biogenesis,17,19 dis-
rupting an intermolecular interaction of NPM1 that
sequesters it in the nucleolus,18 altering biophysical
properties of nucleic acids,34,35 or globally perturbing
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integrity. More work is needed to understand this
fascinating biological stress process and to define the
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge
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(PDF)
Supplementary NMR Spectra (PDF)
AUTHOR INFORMATION
(12)
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Corresponding Author
*derose@uoregon.edu
Author Contributions
#Co-first authors
Funding Sources
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No competing financial interests have been declared.
Saunders, A. M.; DeRose, V. J. Beyond Mg2+: Functional
Interactions between RNA and Transition Metals. Curr.
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This work was supported by the National Science Foun-
dation [CHE1710721 to VJD], the NIH [T32 GM007759-
29 to ECS] and used the Extreme Science and Engineer-
ing Discovery Environment (XSEDE), which is sup-
ported by National Science Foundation [ACI-1548562].
Computations were also performed on the PICS Coeus
high performance computer, which is supported by the
National Science Foundation [1624776]. This work is
also supported by the Department of Chemistry and
Biochemistry, Department of Biology, Institute of Mo-
lecular Biology and the Material Science Institute at the
University of Oregon.
Sutton, E. C.; McDevitt, C. E.; Yglesias, M. V.; Cunningham,
R. M.; DeRose, V. J. Tracking the Cellular Targets of Plati-
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Nucleolar stress is induced by oxaliplatin and a subclass of oxaliplatin derivatives
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