biomedicines

Article
Targeting RNA Polymerase I Transcription Activity in
Osteosarcoma: Pre-Clinical Molecular and Animal
Treatment Studies
Chang-Won Kang 1 , Anneke C. Blackburn 1 , Amos Hong Pheng Loh 2 , Kuick Chick Hong 3 , Jian Yuan Goh 3 ,
Nadine Hein 1 , Denis Drygin 4 , Chris R. Parish 1 , Ross D. Hannan 1,5,6,7 , Katherine M. Hannan 1,5,†
and Lucy A. Coupland 1, *,†

1 The Division of Genome Science and Cancer, The John Curtin School of Medical Research, The Australian
National University, Acton, Canberra 2601, Australia; chang-won.kang@anu.edu.au (C.-W.K.);
kate.hannan@anu.edu.au (K.M.H.)
2 VIVA-KKH Paediatric Brain and Solid Tumour Programme, Children’s Blood and Cancer Centre,
KK Women’s and Children’s Hospital, Singapore 229899, Singapore
3 Department of Pathology and Laboratory Medicine, KK Women’s and Children’s Hospital,
Singapore 229899, Singapore
4 Regulus Therapeutics, 4224 Campus Point C, San Diego, CA 92121, USA
5 Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville 3010, Australia
6 Department of Biochemistry and Molecular Biology, Monash University, Clayton 3800, Australia
7 School of Biomedical Sciences, University of Queensland, St. Lucia 4067, Australia
* Correspondence: lucy.coupland@anu.edu.au
† Co-Last Authors.

Abstract: The survival rate of patients with osteosarcoma (OS) has not improved over the last 30 years.
Citation: Kang, C.-W.; Blackburn,
Mutations in the genes TP53, RB1 and c-Myc frequently occur in OS and enhance RNA Polymerase I
A.C.; Loh, A.H.P.; Hong, K.C.; Goh,
(Pol I) activity, thus supporting uncontrolled cancer cell proliferation. We therefore hypothesised that
J.Y.; Hein, N.; Drygin, D.; Parish, C.R.;
Hannan, R.D.; Hannan, K.M.; et al.
Pol I inhibition may be an effective therapeutic strategy for this aggressive cancer. The Pol I inhibitor
Targeting RNA Polymerase I CX-5461 has demonstrated therapeutic efficacy in different cancers in pre-clinical and phase I clinical
Transcription Activity in trials; thus, the effects were determined on ten human OS cell lines. Following characterisation using
Osteosarcoma: Pre-Clinical genome profiling and Western blotting, RNA Pol I activity, cell proliferation and cell cycle progression
Molecular and Animal Treatment were evaluated in vitro, and the growth of TP53 wild-type and mutant tumours was measured in
Studies. Biomedicines 2023, 11, 1133. a murine allograft model and in two human xenograft OS models. CX-5461 treatment resulted in
https://doi.org/10.3390/ reduced ribosomal DNA (rDNA) transcription and Growth 2 (G2)-phase cell cycle arrest in all OS
biomedicines11041133 cell lines. Additionally, tumour growth in all allograft and xenograft OS models was effectively
Academic Editors: Bruno suppressed without apparent toxicity. Our study demonstrates the efficacy of Pol I inhibition against
Kaufmann Robbs, Fernando OS with varying genetic alterations. This study provides pre-clinical evidence to support this novel
de Carvalho da Silva and Gabriela therapeutic approach in OS.
Nestal De Moraes
Keywords: osteosarcoma; a small molecule inhibitor; RNA polymerase I transcription; CX-5461
Received: 11 March 2023
Revised: 1 April 2023
Accepted: 4 April 2023
Published: 9 April 2023
1. Introduction
Osteosarcoma (OS) is the most frequently occurring primary malignant bone tumour
in children and young adults, with the highest peak incidence during the second decade
Copyright: © 2023 by the authors. of life [1]. Currently, surgical resection with combination chemotherapy consisting of
Licensee MDPI, Basel, Switzerland. cisplatin, doxorubicin and high-dose methotrexate is used as the standard treatment for
This article is an open access article
OS and has increased the 5-year overall survival rate to 60–70% in patients with localised
distributed under the terms and
disease [2]. However, 30% of OS patients do not respond to chemotherapy due to chemo-
conditions of the Creative Commons
resistance, and the outcomes of OS patients with metastatic disease are lower than 20%
Attribution (CC BY) license (https://
at 5 years [3–5]. In addition, recent international clinical trials with more targeted thera-
creativecommons.org/licenses/by/
pies, such as trastuzumab in combination with methotrexate, doxorubicin and cisplatin
4.0/).

Biomedicines 2023, 11, 1133. https://doi.org/10.3390/biomedicines11041133 https://www.mdpi.com/journal/biomedicines

Biomedicines 2023, 11, 1133 2 of 14

(MAP) [6], MAP plus pegylated interferon alpha-2b (IFN- α-2b) [7], or MAP with ifosfamide
and etoposide [8], in OS patients did not improve outcomes. Despite many studies, the
overall survival rate in OS patients has plateaued for three decades; hence, new effective
therapeutic treatments for improving OS outcomes are urgently needed.
Dysregulated cell growth is one of the representative hallmarks of cancer [9] and ac-
companies increased rates of protein synthesis and ribosome biogenesis in cancer cells [10].
RNA Polymerase I (Pol I) plays a role as a rate-limiting factor in ribosome biogenesis via
transcription of the 47S precursor ribosomal RNA (47S rRNA), which is processed into
mature 18S, 5.8S and 28S, the nucleic acid backbone of the ribosome [11–13]. In cancer
cells, impaired signalling pathways (PI3K/Akt and Ras/MAPK) and genetic mutations in
oncogenes (c-Myc and mTOR) and tumour suppressors (TP53, RB1 and ATRX) promote Pol
I transcription activity [12,14,15]. In combination, these findings led to the proposal that
Pol I transcription activity suppression could be an effective therapy against cancers and to
the development of new drugs inhibiting Pol I transcription.
CX-5461, the first-in-class small molecule inhibitor of Pol I-mediated transcription,
suppresses rRNA transcription by preventing the binding of the Pol I-specific transcription
initiation factor, SL-1, to the rDNA promoter region [16,17]. CX-5461 exhibits anti-tumour
activity in the low nanomolar range with higher than 200-fold selectivity for Pol I tran-
scription inhibition compared to Pol-II-mediated transcription [16,18]. Multiple pre-clinical
studies have demonstrated the therapeutic efficacy of CX-5461 in several cancers, in-
cluding B-cell lymphoma [18,19], acute myeloid leukaemia [11], multiple myeloma [20],
prostate cancer [21], breast cancer [22], neuroblastoma [23] and ovarian cancer [24]. Further-
more, CX-5461 has demonstrated single-agent anti-tumour efficacy and was well tolerated
in patients with haematological malignancies in phase I clinical trials. Palmar-plantar
erythrodysesthesia was the only dose-limiting toxicity reported, and photosensitivity
was a dose-independent adverse effect that could be managed by limiting sun expo-
sure [17]. Other clinical trials of CX-5461 in solid tumours are ongoing (NCT02719977 and
NCT04890613).
Recent studies in OS indicate that Pol I transcription activity is very likely to be
dysregulated in OS. c-Myc protein, a key upstream regulator of ribosome biogenesis [25],
is frequently overexpressed in OS [26], and the Pol I transcription-regulatory genes TP53
and RB1 are highly mutated [27–30]. Furthermore, enlarged nucleoli, a classic hallmark
of elevated rDNA transcription activity, have also been described in human OS [31]. A
previous study of RNA Pol I inhibition in osteosarcoma obtained relatively high 50%
growth inhibitory concentration (GIC50 ) values for CX-5461 in two OS cell lines, and a
high concentration of CX-5461 was used in cell cycle and autophagy studies [32]. These
results provided preliminary evidence that inhibiting Pol I transcription using CX-5461
may be an effective therapy against OS. To provide greater justification for the clinical
evaluation of CX-5461 in OS, this study aimed to undertake a comprehensive evaluation
of the therapeutic efficacy of CX-5461 against OS. To do so, ten human OS cell lines were
analysed for alterations in cancer-associated genes and the expression of key proteins
involved in Pol I regulation. The concentrations of CX-5461 required to impact Pol I
transcription, cell proliferation and cell cycle progression were determined. Additionally,
the in vivo efficacy of CX-5461 was assessed in three murine models: (i) a TP53 mutant
human OS xenograft, (ii) a TP53 wild-type human OS xenograft and (iii) a TP53 wild-type
murine allograft.

2. Materials and Methods

2.1. Compound
CX-5461 was purchased from SYNkinase (Parkville, Vic, Australia), and 10 mM stocks
of CX-5461 were prepared in 50 mM NaH2 PO4 (pH4.5) and diluted in growth media
immediately prior to use in vitro. For the in vivo studies, 30 mg/kg of CX-5451 was
prepared in 50 mM NaH2 PO4 (pH4.5).
Biomedicines 2023, 11, 1133 3 of 14

2.2. Cell Culture and Media

Five commercially available human osteosarcoma cell lines, HOS, 143B, MNNG/HOS,
U-2-OS and SJSA-1, were purchased from American Tissue Culture Collection (ATCC® ,
Manassas, VA, USA), and a further five patient-derived osteosarcoma cell lines, KOS-1,
KOS-2, KOS-3, KOS-6 and KOS-7, were kindly provided by HS KIM from Seoul National
University Hospital [33]. Human foreskin fibroblast cells (HF) were purchased from Lonza
(Basel, Switzerland). HOS, 143B and MNNG/HOS cells were cultured with minimum es-
sential medium (MEM) containing 10% foetal bovine serum (FBS) (Sigma-Aldrich, St Louis,
MA, USA), 2 mM L-glutamine (Gibco, Waltham, MA, USA), 1 mM sodium pyruvate (Gibco,
Waltham, MA, USA) and 1% Penicillin/Streptomycin/Neomycin (PSN) (Gibco, Waltham,
MA, USA). U-2-OS cells were cultured with McCoy’s 5A medium (Gibco, Waltham, MA,
USA) containing 10% FBS and 1% PSN. SJSA-1 cells were cultured with RPMI1640 contain-
ing 10% FBS, 1% PSN and 2 mg/mL of d-glucose (Gibco, Waltham, MA, USA). All five
KOS cell lines were cultured with Dulbecco’s modified Eagle medium (DMEM) containing
10% FBS, 4 mM L-glutamine, 1% PSN and 1 mM sodium pyruvate. HF cells were cultured
with DMEM containing 10% FBS, 8 mM L-glutamine and 1% PSN. All cells were cultured
in a humidified cell culture incubator at 37 ◦ C containing 5% CO2 .

2.3. Genome Profiling of Cell Line

Single-nucleotide variants (SNVs) and copy number variants (CNVs) in ten OS cell
lines were analysed using The AmpliseqTM Cancer Childhood Panel DNA assay (Illumina,
San Diego, CA, USA). The assay was conducted following the manufacturer’s instructions
(Supplementary Methods).

2.4. IncuCyte-Based Cell Proliferation Assay

The proliferation of human OS cells (143B: 4.0 × 102 ; HOS: 6.0 × 102 ; MNNG/HOS:
6.0 × 102 ; U-2-OS: 1.0 × 103 ; SJSA-1: 1.0 × 103 ; KOS-1: 1.0 × 103 ; KOS-2: 1.0 × 103 ; KOS-3:
1.0 × 103 ; KOS-6: 1.0 × 103 ; and KOS-7: 1.0 × 103 ) and murine OS cells (2.0 × 103 ) following
exposure to fresh medium containing vehicle or various concentrations of CX-5461 for 72 h
was undertaken as previously described [34]. The dose–response curve and the 50% growth
inhibitory concentration (GIC50 ) were calculated using Graph-Pad Prism (version 9.1.0).

2.5. Western Blotting

The protein levels of p53, MDM2, c-Myc and RB1 were assessed in the ten OS cell lines
(1.0 × 106 ) and human fibroblast cells (1.0 × 106 ) as previously described [34] using primary
and secondary antibodies (Supplementary Table S2) and β-actin as a loading control.

2.6. Quantitative Real-Time PCR for Assessment of rDNA Transcription

rDNA transcription in 1.0 × 105 cells of all OS cell lines (HOS, 143B, MNNG/HOS,
SJSA-1, U-2-OS, KOS-3, KOS-7 and K7M2) was estimated by quantitative real-time PCR
(qRT-PCR) as previously described [19,34]. Briefly, cells were incubated with medium
containing vehicle or various concentrations of CX-5461 for 1 h, following which total RNA
was extracted and then quantified. RNA (500 ng) was treated with DNase and incubated
in a T100TM Thermocycler (Bio-Rad, Hercules, CA, USA), and then cDNA synthesis was
performed with SuperScriptTM IV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA),
Hexameric Random Primers (Promega, Madison, WI, USA) and dNTPs (Invitrogen, Carls-
bad, CA, USA) in a T100TM Thermocycler. All synthesised cDNA samples were loaded
into 96-well PCR plates with SYBRTM Green master mix, primers that targeted the external
transcribed spacer-2 (ETS-2) of the pre-47S ribosomal RNA and the housekeeping gene,
beta-2-microglobulin (β2M) (Supplementary Table S4), and real-time (RT) PCR was per-
formed on a StepOneTM Plus Real-Time PCR System (Applied Biosystems, Foster City, CA,
USA). All settings for cDNA synthesis and RT-PCR are described in Supplementary Table
S3. The dose–response curve and the 50% transcription inhibitory concentration (tIC50 )
were calculated using Graph-Pad Prism (version 9.1.0).
Biomedicines 2023, 11, 1133 4 of 14

2.7. BrdU and PI Staining for Cell Cycle Analysis

All OS cell lines were analysed for cell cycle progression using Bromodeoxyuridine
(BrdU) and Propidium iodide (PI) staining as previously described [34]. Briefly, OS cells
were incubated for 72 h with medium containing vehicle or GIC50 CX-5461 concentrations
(determined from the cell proliferation assay), then labelled with 10 µM BrdU (Sigma-
Aldrich, St. Louis, MO, USA), harvested and stained with PI. Flow cytometry (Becton
Dickinson LSR II) was used to assess the level of staining, and FlowJo software (version 10.0)
was used for data analysis.

2.8. In Vivo Mouse Models

All in vivo experiments were conducted following approval from the Australian
National University Animal Experimentation Ethics Committee (A2017/16 and A2020/36).
Rag 2 knockout (KO) mice were obtained at 6 to 8 weeks of age from the Animal Resource
Centre (Perth, Australia). 143B-Luc cells (1 × 106 ) in 50 µL of Hank’s Balanced Salt Solution
(HBSS) (Sigma-Aldrich, St Louis, MO, USA) or SJSA-1-Luc cells (5 × 106 ) in 50 µL of
HBSS containing 50% (v/v) Matrigel (Corning, NY, USA) were injected subcutaneously
into the right flanks of mice. For the allograft mouse model, K7M2 cells (1 × 106 ) in
50 µL of HBSS were injected subcutaneously into BALB/c wild-type mice on the right
flank. Tumour-bearing mice were monitored daily. Once the average tumour volume
reached 30~50 mm3 , mice were randomised to receive either CX-5461 or vehicle. Thrice
(3 times) weekly for 3 weeks, 30 mg/kg of CX-5461 in 50 mM NaH2 PO4 (pH4.5) or the
corresponding vehicle (50:50 mixture of 50 mM NaH2 PO4 and 50 mM Citric Acid) was
administered via oral gavage. The experiments were terminated 15 days following the start
of drug administration or prior if mice reached the ethical endpoint.

2.9. Statistical Analysis

Statistical analyses were performed using GraphPad Prism software, version 9.1.0.
The dose–response curve fit and 50% inhibitory values were obtained using non-linear
regression. Comparisons of GIC50 and tIC50 between mutant p53 and wild-type p53 OS
cell lines were conducted using a Mann–Whitney U test. Statistical significance between
two groups (vehicle and CX-5461-treated groups) was assessed using two-way ANOVA.

3. Results
3.1. CX-5461 Inhibits OS Cell Proliferation
In our preliminary characterisation analysis using genome profiling and Western
blotting, we confirmed that genes and proteins associated with RNA Pol I activity were
mutated and dysregulated, respectively, in OS cell lines (Supplementary Figures S1 and S2
and Table S1), thus suggesting that CX-5461 may have anti-tumour effects on OS. Consistent
with our expectations, CX-5461 exhibited a dose-dependent reduction in cell density (%
of confluency) following 72 h of treatment (Figure 1A,B). Mutations in TP53 are reported
to confer chemo-resistance and a poor prognosis in OS patients [35]. CX-5461 effectively
suppressed cell proliferation in both normal p53 (wild-type TP53 gene and normal p53
protein expression) (Figure 1A) and abnormal p53 (mutated TP53 gene or dysregulated
p53 protein expression) (Figure 1B) OS cell lines with low nanomolar ranges of 50% GIC50
(p53 abnormal: HOS: 159 nM; 143B: 63 nM; MNNG/HOS: 77 nM; KOS-7: 36 nM; p53 normal:
U-2-OS: 110 nM; SJSA-1: 188 nM; KOS-1: 173 nM; KOS-2: 113 nM; KOS-3: 33 nM; and
KOS-6: 139 nM) (Figure 1C). There was a non-significant trend for normal p53 cell lines to
have higher GIC50 values than abnormal p53 cell lines. These results suggest that CX-5461
has anti-tumour effects on both TP53 mutant and wild-type OS tumours.
 GIC50 (p53 abnormal: HOS: 159 nM; 143B: 63 nM; MNNG/HOS: 77 nM; KOS-7: 36 nM; p53
normal: U-2-OS: 110 nM; SJSA-1: 188 nM; KOS-1: 173 nM; KOS-2: 113 nM; KOS-3: 33 nM;
and KOS-6: 139 nM) (Figure 1C). There was a non-significant trend for normal p53 cell
Biomedicines 2023, 11, 1133 5 of 14
lines to have higher GIC50 values than abnormal p53 cell lines. These results suggest that
CX-5461 has anti-tumour effects on both TP53 mutant and wild-type OS tumours.

KOS-1
KOS-2
120
KOS-3
Confluence (% of Vehicle)

100 KOS-6
U-2-OS
80 SJSA-1
P=ns
60
200
40

CX-5461 GIC50 (nM)

20 150

0
-10 -9 -8 -7 -6 -5 100
[Concentration, M]

HOS
50
143B
120 MNNG/HOS
0
Confluence (% of Vehicle)

KOS-7
100

3
p5

p5
al

al
80

rm
or

no
N

Ab
60

40

20

0
-10 -9 -8 -7 -6 -5
[Concentration, M]

Figure1.1.CX-5461
Figure CX-5461inhibits
inhibits human
human OSOS cell
cell proliferation
proliferationirrespective
irrespective ofof
p53
p53status. (A)(A)
status. Normal
Normal p53p53
and (B) abnormal p53 osteosarcoma cell lines were treated with the indicated concentrations
and (B) abnormal p53 osteosarcoma cell lines were treated with the indicated concentrations of of CX-
5461 or vehicle (50 mM NaH2PO4 (pH 4.5)) for 72 h and assessed for proliferation using the In-
CX-5461®or vehicle (50 mM NaH2 PO4 (pH 4.5)) for 72 h and assessed for proliferation using the
cuCyte ®ZOOM Live Cell Imaging System. Dose–response curves and 50% growth inhibitory con-
IncuCyte
centrationsZOOM
(GIC50) Live
were Cell Imaging
calculated usingSystem. Dose–response
non-linear curvesmean
regression analysis, and 50% growth
+/− SEM of n =inhibitory
3 bio-
concentrations (GIC
logical replicates. ) wereofcalculated
(C)50Effect p53 status onusing
GICnon-linear
50 of CX-5461 regression analysis,
with p53 status mean +/
determined − SEM
from ge- of
n nomic
= 3 biological
profilingreplicates. (C) Effect ofTable
data (Supplementary p53 status
S1) andon GIC
p53 50 of CX-5461
protein with
expression p53instatus
levels determined
Western blots
(Supplementary
from Figuredata
genomic profiling S1). (Supplementary
Normal p53: TP53Table wildS1)type with
and p53the normal
protein level of p53
expression protein
levels ex-
in Western
pression;
blots abnormal p53:
(Supplementary TP53S1).
Figure mutant or a dysregulated
Normal p53: TP53 wild leveltype
of p53 protein
with expression.
the normal levelEach symbol
of p53 protein
represents the mean value of n = 3. Statistical analysis was conducted using a Mann–Whitney U test.
expression; abnormal p53: TP53 mutant or a dysregulated level of p53 protein expression. Each
symbol represents the mean value of n = 3. Statistical analysis was conducted using a Mann–Whitney
3.2. CX-5461 Shows on-Target Activity in OS Cells
U test.

3.2. CX-5461 Shows on-Target Activity in OS Cells

CX-5461 was designed to inhibit Pol I transcription activity by disrupting the tran-
scription initiation complex on the RNA Pol I promoter region, a fundamental component
for Pol I transcription [12]. The on-target activity and mechanism of action of CX-5461 have
been demonstrated in human cancers in pre-clinical and clinical studies [16,17]. To estimate
the on-target activity of CX-5461 on rDNA transcription in OS cell lines, the abundance
of a rapidly processed region of the 47S pre-ribosomal RNA [12] was investigated using
qRT-PCR with CX-5461 treatment. The rDNA transcription rate was found to be reduced
 CX-5461 was designed to inhibit Pol I transcription activity by disrupting the tran-
scription initiation complex on the RNA Pol I promoter region, a fundamental component
for Pol I transcription [12]. The on-target activity and mechanism of action of CX-5461
have been demonstrated in human cancers in pre-clinical and clinical studies [16,17]. To
estimate the on-target activity of CX-5461 on rDNA transcription in OS cell lines, the abun-
Biomedicines 2023, 11, 1133 dance of a rapidly processed region of the 47S pre-ribosomal RNA [12] was investigated 6 of 14
using qRT-PCR with CX-5461 treatment. The rDNA transcription rate was found to be
reduced by CX-5461 in a dose-dependent manner within 1 h of treatment in all OS cell
bylines testedin
CX-5461 (Figure 2A). Except for
a dose-dependent in the KOS-3
manner within cell
1 h line, CX-5461 in
of treatment showed
all OSacell
similar
lines50%
tested
transcription inhibitory concentration (tIC 50) range in normal p53 (U-2-OS: 114 nM; SJSA-
(Figure 2A). Except for in the KOS-3 cell line, CX-5461 showed a similar 50% transcription
1: 171 nM;concentration
inhibitory KOS-3: 752 nM) and)abnormal
(tIC p53 OS cell lines (HOS: 221 nM; 143B: 275 nM;
50 range in normal p53 (U-2-OS: 114 nM; SJSA-1: 171 nM;
MNNG/HOS: 265 nM; KOS-7: 207 nM) (Figure 2B). In the genomic study of KOS-3, no
KOS-3: 752 nM) and abnormal p53 OS cell lines (HOS: 221 nM; 143B: 275 nM; MNNG/HOS:
mutations in genes associated with Pol I transcription regulation were detected, which
265 nM; KOS-7: 207 nM) (Figure 2B). In the genomic study of KOS-3, no mutations in
may explain the relative resistance to Pol I inhibition. The results in this section demon-
genes associated with Pol I transcription regulation were detected, which may explain the
strate that CX-5461 effectively inhibits its primary target, RNA Pol I, regardless of p53
relative resistance to Pol I inhibition. The results in this section demonstrate that CX-5461
status.
effectively inhibits its primary target, RNA Pol I, regardless of p53 status.

Figure2.2. CX-5461
Figure CX-5461 suppresses
suppressesrDNA rDNA transcription in p53
transcription normal
in p53 and abnormal
normal humanhuman
and abnormal OS cells.
OS(A)
cells.
ATCC
(A) ATCC and KOS
and KOS(KOS-3
(KOS-3 and KOS-7)
and osteosarcoma
KOS-7) osteosarcomacellcell
lines were
lines treated
were with
treated CX-5461
with or vehicle
CX-5461 or vehicle
(50 mM NaH2PO4 (pH4.5)) for 1 h, and the rDNA transcription rate was determined by qRT-PCR
(50 mM NaH2 PO4 (pH4.5)) for 1 h, and the rDNA transcription rate was determined by qRT-PCR
using the results of the external transcribed spacer (ETS) normalised to β2 microglobulin (β2M). The
using the resultscurve
dose–response of theand
external transcribed
tIC50 were spacer
estimated (ETS)
using normalised
non-linear to β2 analysis,
regression microglobulin (β2M).
mean +/− SEMThe
of n = 3 biological replicates. (B) Effect of p53 status on tIC50 of CX-5461. Each symbol represents−
dose–response curve and tIC 50 were estimated using non-linear regression analysis, mean +/ SEM
the
n = 3value
ofmean biological
of n =replicates. (B)analysis
3. Statistical Effect of p53
was status onusing
conducted tIC50 aofMann–Whitney
CX-5461. EachUsymbol
test. represents the
mean value of n = 3. Statistical analysis was conducted using a Mann–Whitney U test.
3.3. CX-5461 Leads to G2-Phase Cell Cycle Arrest
3.3. CX-5461 Leads to G2-Phase Cell Cycle Arrest
HOS, 143B, MNNS/HOS and KOS-7 were identified as abnormal p53 (p53 mutant)
cell HOS, 143B,
lines, and MNNS/HOS
U-2-OS, andKOS-3
SJSA-1 and KOS-7were
wereidentified
identified asas abnormal
normal p53 wild-type)
p53 (p53 (p53 mutant)
cell
celllines,
lines and U-2-OS, SJSA-1
(Supplementary and
Figure S1KOS-3 wereS1).
and Table identified as normal
Previously, p53 reported
it has been (p53 wild-type)
that
cell lines (Supplementary
CX-5461 treatment leads toFigure
varyingS1consequences
and Table S1).in Previously, it has been
cell cycle progression reported
that correlatethat
CX-5461
with p53treatment leads
status: (i) p53 to varying
wild-type consequences
cancers: in cell
G1-phase cell cyclecycle
arrestprogression thatmutant
[19,36]; (ii) p53 correlate
with p53 status:
cancers: G2-phase(i) p53
cell wild-type cancers:
cycle arrest [36]. ToG1-phase
determine cellthe
cycle arrest
effects of [19,36];
CX-5461(ii) onp53
OSmutant
cell
cancers: G2-phase cell
cycle progression, a cycle
BrdUarrest
and [36]. To determine
PI staining assay thewaseffects of CX-5461
performed. The on OS cell
HOS, cycle
143B,
progression,
MNNG/HOS, a BrdU
U-2-OS,and PI staining
SJSA-1, KOS-3assay was performed.
and KOS-7 Thetreated
cell lines were HOS, 143B, MNNG/HOS,
with the GIC50 of
U-2-OS, SJSA-1, KOS-3 and KOS-7 cell lines were treated with the GIC50 of CX-5461 for 72 h
and analysed by flow cytometry. CX-5461 treatment was found to induce G2-phase cell
cycle arrest in all OS cell lines analysed, both p53 normal and abnormal cell lines (Figure 3).
Biomedicines 2023, 11, x FOR PEER REVIEW 7 of 14

CX-5461 for 72 h and analysed by flow cytometry. CX-5461 treatment was found to induce
Biomedicines 2023, 11, 1133 7 of
G2-phase cell cycle arrest in all OS cell lines analysed, both p53 normal and abnormal 14
cell
lines (Figure 3).

HOS 143B MNNG/HOS

(Abnormal p53) (Abnormal p53) (Abnormal p53)
120 120 120
100 100 100

% of Cells
80

% of Cells
80
% of Cells

80
60 60 60
40 40 40
20 20 20
0 0 0

61

61
e

61

cl

cl
cl

54

54
hi
54

hi
hi

Ve

Ve
X-
Ve

X-
X-

C
C

U-2-OS SJSA-1 KOS-3 KOS-7

(Normal p53) (Normal p53) (Normal p53) (Abnormal p53)
120 120 120 120
100 100 100 100
80
% of Cells

% of Cells

80
% of Cells

80

% of Cells
80
60 60 60 60
40 40 40 40
20 20 20 20
0 0 0 0
e

61

61
e

61

le

61
cl

cl
cl

54

ic
hi

54
54

hi
hi

54
h
Ve

Ve
X-
Ve

X-
X-

Ve

X-
C

C
C

C
CX-5461 induces
Figure 3. CX-5461 induces G2-phase
G2-phase cell cell cycle arrest in human OS cells. (A–G)
(A–G) ATCC and KOS
osteosarcoma cell
cell lines
lineswere
weretreated
treatedwithwiththe
therespective
respectiveGIC 50 CX-5461
GIC dosedose
50 CX-5461 (determined
(determinedfromfrom
Fig-
ure 2) or
Figure vehicle
2) or (50(50
vehicle mMmMNaH
NaH 2PO
2
4 (pH4.5))
PO 4 (pH4.5))for 72
for h,
72 and
h, the
and theproportion
proportionof
ofcells
cellsin
in each
each cell
cell cycle
cycle
phase was
phase was determined
determinedusing
usingBrdU/PI
BrdU/PI staining
staining and
andflow
flowcytometric
cytometricanalysis.
analysis.Mean
Mean+/ +/−
− SD
SD of
of nn== 22
biological replicates. G1/G0 = quiescent and growth phases not involving proliferation; S = DNA
biological replicates. G1/G0 = quiescent and growth phases not involving proliferation; S = DNA
synthesis phase; and G2/M = growth and preparation for mitosis and mitosis phases.
synthesis phase; and G2/M = growth and preparation for mitosis and mitosis phases.

3.4. CX-5461 Reduces Tumour Growth in

Tumour Growth in Human
Human OS OS Xenografts
Xenografts
The anti-tumour activity of CX-5461 was investigated in two human OS xenograft
mouse models in Rag 2 knockout (KO) mice using 143B (p53 mutant-type) and SJSA-1
(Figure 4).
(p53 wild-type) cell lines (Figure 4). Each experiment was repeated to demonstrate the
reproducibility of the results. CX-5461 effectively suppressed 143B tumour growth, and
this effect was significant (p < 0.05)
0.05) after
after the
the first
first week
week (Day
(Day 8)
8) of
of drug
drug dosing
dosing (Figure
(Figure 4B).
4B).
Similarly, CX-5461 showed tumour-growth-inhibitory activity on SJSA-1 tumours, and
the effect was significant on Day 11 of treatment (Figure 4C). CX-5461 was well tolerated
without excessive reductions
reductions inin body
body weight
weight (Figure
(Figure 4D,E).
4D,E).
 Biomedicines
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1133x FOR PEER REVIEW 8 of 814of 14

Figure4.4.CX-5461
Figure CX-5461reduces
reduces tumour
tumour growth
growthin inhuman
humanOS OSxenograft
xenograft mouse
mouse models. (A)(A)
models. Schematic of of
Schematic
human OS xenograft mouse model: OS cells were injected subcutaneously into the right flanks of
human OS xenograft mouse model: OS cells were injected subcutaneously into the right flanks of
male and female Rag2 KO mice; then, when tumours could be measured using digital callipers, mice
male and female Rag2 KO mice; then, when tumours could be measured using digital callipers, mice
were randomised (ensuring equal distribution of sex and tumour size) to vehicle (50 mM NaH2PO4
were randomised
(pH4.5)) (ensuring
or CX-5461 equal
(30 mg/kg) distribution
group of sex
thrice weekly forand
twotumour size)
weeks. (B) to vehicle
143B-Luc OS(50cellsmM NaH2 PO4
(abnormal
(pH4.5)) or CX-5461 (30 mg/kg) group thrice weekly for two weeks. (B) 143B-Luc OS
p53; 1 × 10 cells in 50 µL of HBSS) or (C) SJSA-1-Luc OS cells (normal p53; 3 × 10 in 50 µL of HBSS
6 6 cells (abnormal
p53; 1 × 10650%
containing cellsMatrigel)
in 50 µLwere injected.
of HBSS) Tumour
or (C) volumeOS
SJSA-1-Luc was monitored
cells (normal and 3 × 106 in
p53;expressed in50
each
µL of
mouse as the fold change in the tumour size at the start of treatment, with the
HBSS containing 50% Matrigel) were injected. Tumour volume was monitored and expressed in average of each treat-
ment group depicted. (D,E) The weight of each mouse was monitored during the treatment period,
each mouse as the fold change in the tumour size at the start of treatment, with the average of each
with a 20% reduction in body weight being the ethical end point (dotted line), which was not
treatment group+/−
reached. Mean depicted. (D,E) The
SEM of biological weight as
replicates ofindicated.
each mouse was monitored
Statistical during
analysis was the treatment
performed using
period,
two-waywith a 20% reduction
ANOVA, in body
* p < 0.05, **** weight being the ethical end point (dotted line), which was
p < 0.0001.
not reached. Mean +/− SEM of biological replicates as indicated. Statistical analysis was performed
using two-way ANOVA, * p < 0.05, **** p < 0.0001.
Biomedicines 2023,
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2023, 1133x FOR PEER REVIEW 9 of 914of 14

3.5.
3.5.CX-5461
CX-5461Demonstrates
Demonstrates Therapeutic Efficacyininan
Therapeutic Efficacy anImmunocompetent
Immunocompetent Mouse
Mouse Model
Model of OS
of OS
ToToinvestigate whetherCX-5461
investigate whether CX-5461 hadhad
the the
samesame in therapeutic
in vivo vivo therapeutic
efficacyefficacy in the
in the pres-
ence of aoffunctional
presence immune
a functional immunesystem, the anti-tumour
system, effecteffect
the anti-tumour of CX-5461 was evaluated
of CX-5461 in
was evaluated
inananallograft mouse
allograft mousemodel
modelusing the the
using murine OS cell
murine OS line
cell K7M2 (Figure
line K7M2 5). As5).
(Figure seenAsinseen
the in
human
the human OSOSxenograft
xenograftmouse
mouse models,
models, CX-5461
CX-5461effectively
effectivelysuppressed
suppressed K7M2
K7M2 tumour
tumour
growthininBALB/c
growth BALB/c mice andandwas
waswell
welltolerated
toleratedwithout
without significant changes
significant changes in animal body
in animal body
weight(Figure
weight (Figure5B,C).
5B,C). The
The in
in vitro
vitro studies
studiesof ofCX-5461
CX-5461on onthe
theK7M2
K7M2 cell line
cell demonstrated
line demonstrated
thesuppression
the suppression ofof RNA
RNA Pol
Pol II transcription
transcription andandcell
cellproliferation
proliferation via G2-phase
via G2-phase cellcell
cycle
cycle
arrest (Supplementary Figure
arrest (Supplementary Figure S3). S3).

Figure5.5.CX-5461
Figure CX-5461inhibits
inhibits tumour
tumour cell
cellgrowth
growthininan animmune-competent
immune-competent mouse model
mouse of osteosar-
model of osteosar-
coma. (A) Schematic of immune-competent murine model of osteosarcoma: K7M2 murine OS cells
coma. (A) Schematic of immune-competent murine model of osteosarcoma: K7M2 murine OS cells
(1 × 1066 in 50 µL of HBSS) were injected subcutaneously into the right flanks of male and female
× 10 in
(1BALB/c 50 µL
mice; ofwhen
then, HBSS) were injected
tumours could besubcutaneously into the
measured using digital right flanks
callipers, of male
mice were and female
randomised
BALB/c
(ensuringmice; then,
equal when tumours
distribution of sex could be measured
and tumour size) tousing digital
vehicle callipers,
(50 mM NaH2PO mice were randomised
4 (pH4.5)) or CX-
(ensuring
5461 (30 equal
mg/kg)distribution
group thriceof sex and for
weekly tumour size) to(B)
two weeks. vehicle
Tumour(50 mM NaHwas
volume 2 PO (pH4.5))
monitored
4 or
andCX-5461
ex-
pressed
(30 mg/kg) in each
groupmouse
thriceasweekly
the fold-change in the tumour
for two weeks. size atvolume
(B) Tumour the startwas
of treatment,
monitored with
andthe aver-
expressed
inage
eachof mouse
each treatment group depicted.
as the fold-change in the(C) The weight
tumour size atofthe
each mouse
start was monitored
of treatment, during
with the the of
average
treatment period, with a 20% reduction in body weight being the ethical end point (dotted line),
each treatment group depicted. (C) The weight of each mouse was monitored during the treatment
which was not reached. Mean +/− SEM of biological replicates as indicated. Statistical analysis was
period, withusing
performed a 20%two-way
reduction in body
ANOVA; * pweight being
< 0.05, ** the ethical
p < 0.005, **** p <end point (dotted line), which was
0.0001.
not reached. Mean +/− SEM of biological replicates as indicated. Statistical analysis was performed
using two-way ANOVA; * p < 0.05, ** p < 0.005, **** p < 0.0001.
4. Discussion
The advancement of diagnostic and therapeutic strategies for OS has not improved
outcomes over the last 30 years, and chemo-resistance is a major problem. There is, there-
fore, an urgent need for new therapeutic strategies against this disease [1,7,8,37]. Acceler-
ated RNA Pol I transcriptional activity is common to many cancers, including OS; thus,
Biomedicines 2023, 11, 1133 10 of 14

4. Discussion
The advancement of diagnostic and therapeutic strategies for OS has not improved
outcomes over the last 30 years, and chemo-resistance is a major problem. There is,
therefore, an urgent need for new therapeutic strategies against this disease [1,7,8,37].
Accelerated RNA Pol I transcriptional activity is common to many cancers, including OS;
thus, targeting this protein represents a novel therapeutic approach. In this study, we have
(i) determined the CX-5461 GIC50 and tIC50 values for each OS cell line, (ii) demonstrated
the on-target effect of CX-5461 on Pol I activity, (iii) analysed the effect of CX-5461 on cell
division in a range of cell lines and (iv) demonstrated the therapeutic effect of CX-5461
in vivo using three different mouse models.
Alterations in tumour suppressors and oncogenes that occur in many cancers influence
Pol I transcription activity [12,14]. The tumour suppressors p53 and RB1 suppress Pol I
transcription by blocking the assembly of the pre-initiation complex, SL-1 and UBF, on the
rDNA promoter region [38,39]. p53 and RB1 also suppress the transcription of the oncogene
c-Myc, which enhances Pol I transcription via stimulation of UBF and SL-1 binding to the
rDNA promoter region [25,40,41]. Our preliminary characterisation analysis of the OS
cell lines identified molecular alterations that would impact the presence of mutations
in TP53, RB1, TSC1, TSC2 and ATRX and amplifications of c-Myc, MDM2 and CDK4.
Additionally, the dysregulated expression of p53, RB1, c-Myc and MDM2 proteins was
demonstrated, all of which have the potential to impact Pol I activity. These molecular
results are consistent with other genetic studies of OS and highlight the significant diversity
in gene and protein alterations that may lead to OS and enhanced Pol I activity, and they
emphasise the potential for Pol I inhibitors to be effective against the majority of OS cases.
Multiple studies have revealed the mechanism of the therapeutic effect of CX-5461
on human cancers [11,16–18,20,24,36,42,43]. CX-5461 prevents the assembly of the Pol
I-specific pre-initiation complex factor, SL-1, on the rDNA promoter region, leading to the
abrogation of Pol I binding [18]. Consequently, these changes result in anti-tumour effects
through the p53-dependent nucleolar stress response (NSR) and p53-independent NSR
pathways [19,36,44]. In this study, CX-5461 demonstrated anti-cancer activity across the
panel of 10 OS cell lines. CX-5461 was shown to rapidly suppress rDNA transcription, the
primary target, within 1 h of treatment, and subsequently induced anti-proliferative effects
in all OS cell lines, both normal p53 and abnormal p53, through G2-phase cell cycle arrest.
These results extend the evidence of CX-5461’s anti-tumour effects on OS, irrespective of
the p53 status, thus implying the same multi-pathway mechanisms.
In clinical studies, the response to chemotherapy and the survival rate of OS patients
varied according to the TP53 gene mutation status [45,46]. A meta-analysis of eight pub-
lished studies demonstrated that patients with tumours bearing TP53 mutations had a
significantly higher risk of death within 2 years than patients with wild-type TP53 tu-
mours [35]. Our in vivo efficacy studies of CX-5461 showed a significant and reproducible
therapeutic effect on both p53 mutant OS, 143B, and p53 wild-type OS, SJSA-1, tumours.
CX-5461 also exhibited a significant tumour-growth-inhibitory effect in an allograft mouse
model with a functional immune system. A previous study of CX-5461 on human OS
in vitro found that CX-5461 induced anti-tumour activity via the activation of autophagy
involving the mammalian target of rapamycin (mTOR) signalling pathways in the OS cell
lines U-2-OS and MNNG/HOS [32]. The drug doses used in this study were significantly
higher (43-fold) than those used in our studies. CX-5461 was also observed to suppress
U-2-OS tumour growth in an in vivo mouse model.
Previous studies of the anti-proliferative effect of CX-5461 in 50 cancer cell lines and
5 normal cell lines demonstrated a median GIC50 value in the cancer cell lines of 147 nM
(range 3–5500 nM), whereas the 5 normal cell lines all had a GIC50 value of approximately
5000 nM [16]. The GIC50 values obtained for the 10 OS cell lines in this study (33–188 nM),
therefore, closely align with the median values previously reported for other cancer cell
lines. More importantly, these values are well below those required to impact the prolif-
eration of normal cell lines; thus, these doses could be considered selective for OS cells.
Biomedicines 2023, 11, 1133 11 of 14

The lack of obvious side effects in our mouse studies, with a maximum weight loss of
10% measured, reflects the results of early phase trials in humans in which CX-5461 was
well tolerated [17]. Interestingly, the most profound anti-tumour effect of CX-5461 was
seen in the immune-competent mouse model, which also displayed the most rapid tumour
growth and the least weight loss. As immune cells may enhance or inhibit tumour growth,
further studies would be required to determine what effects CX-5461 has on the cells of the
immune system in this model.
As with most chemotherapy agents, resistance to RNA Pol I inhibition may develop as
a result of new mutations or compensatory mechanisms. Combining therapies that target
different pathways increases the treatment effect and decreases the likelihood of resistance
occurring. CX-5461 was successfully combined with the mTORC1 inhibitor everolimus to
demonstrate a synergistic anti-tumour effect in lymphoma-bearing mice [17]. Future trials
in OS could combine CX-5461 with more commonly used agents, such as methotrexate,
doxorubicin and cisplatin.
In conclusion, this study has identified mutations in genes associated with Pol I
activity in 10 OS cell lines and has confirmed the alteration of proteins well recognised to be
involved in the regulation of Pol I transcription. Furthermore, this study has demonstrated
that targeting elevated Pol I transcription in OS shows significant promise as an effective
novel therapeutic strategy. Given the pre-clinical results provided and the safety profile
of CX-5461 already obtained in human clinical studies, CX-5461 should be considered a
potential therapeutic option for OS.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/biomedicines11041133/s1, Figure S1: Western blots of four key
proteins that are known regulator of RNA Pol I activity; Figure S2: Uneditied western blots of four
key proteins that are known regulator of RNA Pol I activity; Figure S3: In vitro antitumour effects of
CX-5461 on K7M2; Table S1: Genetic mutations in OS cell lines; Table S2: List of antibodies; Table S3:
qPCR setting; Table S4: qPCR primer sequences.
Author Contributions: Conceptualisation, C.-W.K., K.M.H. and L.A.C.; methodology, C.-W.K.,
A.H.P.L., K.C.H. and J.Y.G.; validation, C.-W.K. and A.C.B.; formal analysis, C.-W.K., A.C.B., A.H.P.L.,
K.C.H. and J.Y.G.; investigation, C.-W.K., A.H.P.L., K.C.H. and J.Y.G.; resources, C.R.P., R.D.H.,
K.M.H. and L.A.C.; data curation, C.-W.K. and A.C.B.; writing—original draft preparation, C.-W.K.;
writing—review and editing, C.-W.K., A.C.B., N.H., R.D.H., D.D., A.H.P.L., K.M.H. and L.A.C.;
visualisation, C.-W.K.; supervision, K.M.H. and L.A.C.; project administration, K.M.H. and L.A.C.;
funding acquisition, R.D.H., K.M.H. and L.A.C. All authors have read and agreed to the published
version of the manuscript.
Funding: Funding was obtained from the Sarah Grace Sarcoma Foundation, the VIVA Foundation
for Children with Cancer, and a Johanna Sewell Osteosarcoma Research Grant to support this work.
Additionally, a National Health and Medical Research (NHMRC) of Australia Program Grant (APP
No. 1053792) and an NHMRC Principal Research Fellowship (No. 116999) supported this work.
Institutional Review Board Statement: The animal ethics protocol (A2017/16) was approved by the
Animal Experimentation Ethics Committee (AEEC) of the Australian National University.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Thanks to the staff of Imaging and Cytometry and the ACRF Biomolecular
Research Facilities of The John Curtin School of Medical Research, The Australian National University,
for their assistance with equipment and settings.
Conflicts of Interest: The authors declare no competing interest.
Biomedicines 2023, 11, 1133 12 of 14

Abbreviations
Akt AKT serine/threonine kinase 1
ATM Ataxia telangiectasia-mutated kinase
ATR Ataxia telangiectasia and Rad3-related kinase
ATRX Alpha-thalassemia/mental retardation x-linked chromatin remodeller
CDK4 Cyclin-dependent kinase 4
c-Myc Cellular myelocytomatosis
GIC50 50% growth inhibitory concentration
KO Knockout
MAP Methotrexate, doxorubicin and cisplatin
MAPK Mitogen-Activated Protein Kinase
MDM2 E3 ubiquitin-protein ligase MDM2
mTOR Mammalian target of rapamycin
OS Osteosarcoma
p53 Tumour suppressor protein 53
Pol I RNA Polymerase I
PI3K Phosphoinositide 3-kinase
rDNA Ribosomal DNA
RB1 Retinoblastoma 1
SL-1 Selective factor 1
tIC50 50% transcription inhibitory concentration
TP53 p53 gene
TSC Tuberous Sclerosis Complex
UBF Upstream binding factor

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