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· Introduction: – Explained importance of topic and sufficient background to tie together articles

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· Article 2 Summary: – Briefly explained methods, results, conclusions

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Research Article
Nucleolar Division in the Promastigote Stage of
Leishmania major Parasite: A Nop56 Point of View

Tomás Nepomuceno-Mej-a , Luis Enrique Florencio-Mart-nez,
and Santiago Mart-nez-Calvillo

Unidad de Biomedicina, Facultad de Estudios Superiores Iztacala, Universidad Nacional Autónoma de México. Av. de los Barrios 1,
Col. Los Reyes Iztacala, Tlalnepantla, Estado de México, CP 54090, Mexico

Correspondence should be addressed to Tomás Nepomuceno-Mej́ıa; tnepomuceno@unam.mx
and Santiago Mart́ınez-Calvillo; scalv@unam.mx

Received 15 June 2018; Revised 14 August 2018; Accepted 13 September 2018; Published 10 October 2018

Academic Editor: Amogh A. Sahasrabuddhe

Copyright © 2018 Tomás Nepomuceno-Mej́ıa et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

Nucleogenesis is the cellular event responsible for the formation of the new nucleoli at the end of mitosis. This process depends
on the synthesis and processing of ribosomal RNA (rRNA) and, in some eukaryotes, the transfer of nucleolar material contained
in prenucleolar bodies (PNBs) to active transcription sites. The lack of a comprehensive description of the nucleolus throughout
the cell cycle of the human pathogen Leishmania major prompted us to analyze the distribution of nucleolar protein 56 (Nop56)
during interphase and mitosis in the promastigote stage of the parasite. By in silico analysis we show that the orthologue of Nop56
in L. major (LmNop56) contains the three characteristic Nop56 domains and that its predicted three-dimensional structure is also
conserved. Fluorescence microscopy observations indicate that the nucleolar localization of LmNop56 is similar, but not identical,
to that of the nucleolar protein Elp3b. Notably, unlike other nucleolar proteins, LmNop56 remains associated with the nucleolus in
nonproliferative cells. Moreover, epifluorescent images indicate the preservation of the nucleolar structure throughout the closed
nuclear division. Experiments performed with the related parasite Trypanosoma brucei show that nucleolar division is carried out
by an analogous mechanism.

1. Introduction

The cell nucleus contains a collection of nonmembrane-
bound nuclear bodies (NBs) that participate in the regula-
tion of essential functions, such as gene expression [1, 2].
The nucleolus is the most conspicuous NB that is present
throughout the Eukarya domain [3, 4]. The fundamental role
of the nucleolus is to coordinate ribosome biogenesis, an
intricate multistep process that includes the transcription of
ribosomal cistrons (rDNA) by RNA polymerase (RNA Pol) I
and accessory factors, cleavage and chemical modification of
precursor ribosomal RNA (rRNA), and assembly of mature
rRNA species 18S, 5.8S, and 25/28S with numerous proteins
and the 5S rRNA, product of RNA Pol III activity [5, 6].

The nucleolus is a dynamic organelle that is disassembled
and assembled in organisms undergoing an open mitosis,
such as human cells [7, 8]. The nucleolar cycle begins
during the early stages of nuclear division, when several key

nucleolar proteins involved in rDNA transcription and rRNA
processing are negatively modulated by specific phosphoryla-
tion carried out by the cyclin B-dependent kinase 1 pathway
[9–11]. Consequently, the rRNA synthesis is shut down and
the nucleolar structure disappears. While proteins that par-
ticipate in rDNA transcription remain attached to nucleolar
organizer regions (NORs), rRNA processing proteins and
small nucleolar RNAs (snoRNAs) as well as preserved pre-
rRNAs localize to the cytoplasm and progressively accumu-
late along the entire periphery of condensed chromosomes,
forming part of the perichromosomal compartment (PC)
[12–15]. During chromosomal segregation, the components
of PC migrate together with sister chromatids toward the
poles of the mitotic spindle and remain associated with them
until PC fragmentation. After that, the nucleolar material
accumulates in intermediate nuclear structures called prenu-
cleolar bodies (PNBs), before being released into transcrip-
tionally active NORs, which are chromosomal loci where

Hindawi
BioMed Research International
Volume 2018, Article ID 1641839, 11 pages
https://doi.org/10.1155/2018/1641839

http://orcid.org/0000-0002-8722-4765

http://orcid.org/0000-0002-0247-7879

https://creativecommons.org/licenses/by/4.0/

https://doi.org/10.1155/2018/1641839

2 BioMed Research International

the synthesis and processing of rRNA have been reactivated.
Restoration of ribosome biogenesis, close to the end of
mitosis, triggers the nucleolar reassembly, a cellular process
termed nucleogenesis [7, 8, 13, 16–24]. In Saccharomyces
cerevisiae, an organism with closed mitosis, the nucleolus
is located adjacent to the nuclear envelope, opposite to the
spindle pole body. Unlike higher eukaryotes, the nucleolus
persists during mitosis, and the duplicated nucleolus splits
in two in early telophase, adopting symmetrical positions
in mother and daughter nuclei [25, 26]. Given that the
nucleolus is preserved, PNBs may not be formed during yeast
mitosis, but to the best of our knowledge, this issue has not
been addressed. However, as the nucleolus in this organism
disassembles and reassembles during meiosis [26, 27], it is
possible that PNBs might be involved in nucleolar assembly
in this particular process.

Among the large number of components that are part of
the nucleolar proteome, the nucleolar protein 56 (Nop56) is
an essential factor highly conserved from Archaea to human
that is actively involved in the biogenesis of the ribosomal
subunits. It is one of the core elements of box C/D small nucle-
olar ribonucleoprotein particles (snoRNPs), which direct 2󸀠-
O- ribose methylation of specific residues in pre-rRNA [28–
30] and are also involved in the endonucleolytic cleavages
of the 35S rRNA primary transcript [31, 32]. In addition to
Nop56, C/D snoRNPs contain a C/D snoRNA (like U3 or
U14) and three other core proteins: fibrillarin, Nop58, and
Snu13 [33].

In contrast to yeast and higher eukaryotes, little is known
about structure and biogenesis of the nucleolus in the early-
branched protozoan parasite Leishmania, the etiological
agent of leishmaniasis, a significant public health problem
in tropical and subtropical areas of the world. Leishma-
nia is a member of the Trypanosomatidae family, which
includes the pathogen parasites Trypanosoma brucei and
Trypanosoma cruzi. Leishmania develops within phagolyso-
somes of infected macrophages as amastigotes and in the
gut of the sandfly vector as extracellular promastigotes. The
L. major genome possesses only ∼12 copies of the rDNA
unit per haploid genome, located on chromosome 27 as
head-to-tail tandem arrays [34]. Synthesis and processing of
rRNA are necessary steps for nucleolar building around the
rDNA repeats grouped in transcriptionally active NORs. An
ultrastructural analysis performed in L. major promastigotes
showed that this parasite has a central, single, and spherical
electro-dense nucleolus that, apparently, does not contain a
fibrillar center [35].

Since Nop56 is an appropriate protein to investigate the
process of nucleolar division, in this study we identified and
analyzed the cellular location of the Nop56 orthologue in
L. major (LmNop56). Bioinformatics analyses revealed that
LmNop56 contains the three structural and evolutionary
conserved domains and that its predicted three-dimensional
structure is remarkably similar to that of the S. cerevisiae
orthologue. By indirect immunofluorescence we showed that,
in contrast to other nucleolar proteins, LmNop56 remains
located in the nucleolus in aged cells. Moreover, our data
showed that during interphase and closed mitosis LmNop56
persists and, seemingly, remains associated with the nucle-
olus. Interestingly, similar observations were obtained in
procyclic T. brucei parasites.

2. Material and Methods

2.1. In Silico Analysis. Nop56 amino acid sequences of
trypanosomatids, yeast, and human were obtained from
TriTrypDB (http://tritrypdb.org/tritrypdb/) (release 36), S.
cerevisiae genome (https://www.yeastgenome.org), and Uni-
ProtKB (https://www.uniprot.org), respectively. Multiple
sequences alignments were performed with the Clustal Ω
program (http://www.ebi.ac.uk/Tools/msa/clustalo/) and iden-
tical residues were colored manually. LmNop56 secondary
structure determination was done using UCSF Chimera
package (https://www.cgl.ucsf.edu/chimera/) and PSIPRED
Protein Sequence Analysis Workbench (http://bioinf.cs.ucl
.ac.uk/psipred/). Conserved domains were identified by Pfam
(http://pfam.xfam.org), SMART (http://smart.embl-heidel-
berg.de), Prosite (http://prosite.expasy.org), and InterPro
(https://www.ebi.ac.uk/interpro/) web pages. Three-dimen-
sional homology models were obtained with SWISS-MODEL
(https://swissmodel.expasy.org) and UCFS Chimera program
[36] using the structure of S. cerevisiae (SWISS-MODEL
Template Library ID: 5wyj.3.A) as a model.

2.2. Parasites. L. major promastigotes, strain MHOM/IL/81/
Friedlin (LSB-132.1), were grown in BM medium (1×
M199 medium pH 7.2 containing 10% heat-inactivated fetal
bovine serum, 0.25× brain heart infusion, 40 mM HEPES,
0.01 mg/mL hemin, 0.0002% biotin, 100 IU/mL penicillin,
100 g/mL streptomycin, and 1× L-glutamine) at 28∘C and
harvested in the mid logarithmic (Log) or stationary (Sta)
phases, four or seven days after initial inoculation. The L.
major cell line that expresses a PTP-tagged version of the
nucleolar protein Elp3b [37, 38] was maintained in BM
medium with 50𝜇g/mL G418. Procyclic parasites of the
T. brucei strain 29-13 were cultured in SDM-79 medium
supplemented with 10% fetal bovine serum at 28∘C and
harvested in the mid logarithmic phase. Epimastigotes of
T. cruzi CL Brener strain were grown in LIT medium, as
described elsewhere [39].

2.3. Western Blot Analysis. Trypanosomatid total protein
extracts were solubilized in 5×Laemmli’s buffer, fractionated
by 10% SDS-PAGE and blotted onto a PVDF matrix. Western
blot was performed using a polyclonal anti-LmNop56 mice
serum [37] diluted 1:1000 in 2% nonfat dry milk prepared
in phosphate buffered saline (PBS) containing 0.05% Tween-
20. Antibody-antigen complexes were revealed by chemi-
luminescence, utilizing horseradish peroxidase-labeled goat
anti-mouse IgG (BioLegend) and Immobilon� Western kit
(MILLIPORE). An 𝛼/𝛽-tubulin polyclonal antibody (Cell
Signaling Technology) was used as loading control.

2.4. Immunofluorescence Microscopy. Parasites were col-
lected, rinsed twice with PBS, and attached onto poly-L-
lysine-coated glass slides for 20 min at room temperature.
Then, cells were fixed with 4% paraformaldehyde in PBS
for 30 min at 4∘C and permeabilized with 0.1% Triton x-
100 in PBS for 10 min at room temperature. After several
PBS washes, the unspecific binding sites were blocked with
2% bovine serum albumin (BSA) in PBS during 60 min.

http://tritrypdb.org/tritrypdb/

https://www.yeastgenome.org

https://www.uniprot.org

http://www.ebi.ac.uk/Tools/msa/clustalo/

https://www.cgl.ucsf.edu/chimera/

http://bioinf.cs.ucl.ac.uk/psipred/

http://pfam.xfam.org

http://smart.embl-heidelberg.de

http://prosite.expasy.org

https://www.ebi.ac.uk/interpro/

https://swissmodel.expasy.org

BioMed Research International 3

Preimmune or anti-LmNop56 immune sera were diluted
in 1% BSA in PBS and incubated with the samples for
2 hours. After washing, goat anti-mouse IgG (H+L) anti-
body conjugated with Alexa Fluor� 488 dye (Molecular
probes) was used. DNA was counterstained with propid-
ium iodide and parasites were mounted with Vectashield�.
For confocal microscopy, individual optical sections were
obtained using a Carl Zeiss LSM 5 Pascal confocal laser
microscope. Confocal micrographs were analyzed and pre-
pared for presentation using the ImageJ processing pro-
gram (https://imagej.nih.gov/ij/). On the other hand, in
epifluorescence microscopy analysis, preparations of mid
logarithmic and stationary phase parasites were coverslipped
with Vectashield� mounting medium plus 4󸀠,6-diamidino-
2-phenylindole (DAPI; Vector Laboratories Inc.) after anti-
bodies interaction. Visualization of fluorescent signal was
carried out in a Carl Zeiss Axio Vert.A1 epifluorescence
microscope. For double labeling experiments, cells were
incubated overnight with 1% BSA in PBS containing a
mix of anti-LmNop56 mice immune serum with (1) anti-
histone H4 (Abcam), (2) anti-Prot C (for Elp3b-PTP) (Delta
Biolabs), or (3) 𝛼/𝛽-tubulin (Cell Signaling Technology)
rabbit antibodies. LmNop56 was revealed by goat anti-mouse
IgG (H+L) antibody conjugated with Alexa Fluor� 568 dye.
Histone H4, Protein C-tag, and𝛼/𝛽-tubulin were visualized
by goat anti-rabbit IgG (H+L) antibody coupled with Alexa
Fluor� 488 dye. These samples were covered with antifading-
DAPI solution, as described above. Elp3b distribution was
analyzed in an L. major cell line where Elp3b was labeled with
a PTP tag [37], using the anti-Prot C antibody in combination
with an anti-𝛽-tubulin antibody (Thermo Fisher). The Elp3b
recombinant protein was observed using a goat anti-rabbit
IgG (H+L) cross-adsorbed secondary antibody conjugated
with Alexa Fluor� 594 dye. 𝛽-tubulin was visualized with
goat anti-mouse IgG (H+L) antibody conjugated with Alexa
Fluor� 488 dye. Parasite preparations were coverslipped with
Vectashield� mounting medium plus DAPI, as indicated
above. Epifluorescence micrographs were analyzed and pre-
pared for presentation using the ZEN 2012 software (Blue
edition).

3. Results

3.1. LmNop56 Is an Evolutionarily Conserved Protein. In L.
major, Nop56 is a 473 amino acid protein with a predicted
molecular mass of 52.7 kDa, encoded by a single copy gene
(ID: LmjF.10.0210) found on chromosome 10. Sequence anal-
ysis revealed that, like Archaea and eukaryotic orthologues,
LmNop56 contains the three highly conserved domains
termed NOP5NT (residues 5-70), NOSIC (residues 172-224),
and Nop (residues 225-419) (Figure 1(a)). In other organisms,
these domains are essential for the appropriate assembly
and function of the box C/D snoRNPs. Multiple sequence
alignments indicated that LmNop56 is ∼80% identical to
the T. brucei and T. cruzi orthologues, and 46 and 48%
identical to Nop56 from human and yeast, respectively. The
highest degree of primary structure conservation of Nop56
occurs within the Nop motif (Figure 1(a)). Although the
sequence of the NOP5NT domain is the least conserved,

it is predicted to fold into three 𝛽-sheets that are highly
conserved across evolution (Figure 1(a), data not shown).
The rest of LmNop56 mainly folds into 𝛼-helices dispersed
throughout the protein. The three-dimensional structure for
Nop56 from S. cerevisiae was recently obtained by cryo-
electron microscopy, as part of the modeling of the entire
90S small subunit preribosome [40]. Homology modeling
revealed that the hypothetical three-dimensional structure
of LmNop56 (residues 8 to 421) is extensively similar to
the reported yeast model, showing discrete N-terminal
(NOP5NT) and C-terminal (Nop) domains (Figure 1(b)).
The predicted structure for Nop56 from T. brucei is almost
identical to the one obtained for LmNop56 (Figure 1(b)).
Thus, the in silico analysis demonstrated that LmNop56
contains all the sequence and structural features that are
present in Nop56 orthologues in other organisms.

3.2. LmNop56 Is a Nucleolar Component. To determine the
expression of LmNop56 in L. major promastigotes, West-
ern blot analysis was performed with a mouse polyclonal
immune serum raised against the recombinant version of
this protein [37]. A band of ∼53 kDa was observed, which
corresponds to the predicted size of LmNop56 (52.7 kDa)
(Figure 2(a)). Notably, the polyclonal serum also recognized
Nop56 in procyclic forms of T. brucei (54.3 kDa) and epi-
mastigotes of T. cruzi (53.6 kDa) (Figure 2(a)). In order to
determine the subcellular distribution of LmNop56, indirect
immunofluorescence experiments were performed on fixed
and permeabilized promastigotes using the anti-LmNop56
mice serum. Stained parasites were examined by confocal
(Figure 2(b)) or wide-field optical epifluorescent microscopy
(Figures 2(c) and 2(d)). This analysis clearly revealed a green
fluorescent nuclear body located within a nucleoplasm region
weakly stained with nucleic acid dye propidium iodide in
actively replicating parasites, which might correspond to
nucleolus of L. major (Figure 2(b)). A similar localization
was observed by simultaneous labeling of Nop56 and histone
H4 proteins, where the fluorescent red signal of LmNop56 is
present within a specific region of the nucleoplasm (shown in
green; Figure 2(c)). Nuclear and kinetoplast DNA are shown
in blue. The nucleolar position of LmNop56 was confirmed
by colocalization assays with the nucleolar protein Elp3b
(Figure 2(d)). Elp3b is involved in Pol I transcription of rDNA
in T. brucei [41] and colocalizes with 18S rRNA genes and
with 5S rRNA in L. major [37]. While the majority of the
signal overlaps (yellow spots in Figure 2(d)), some differences
were observed in the nucleolar distribution of Nop56 and
Elp3b. Therefore, these results demonstrate that LmNop56 is
a nucleolar protein that partially colocalizes with Elp3b.

3.3. LmNop56 Is Concentrated in the Nucleolus and Additional
Nuclear Regions in Nonproliferative Parasites. To investigate
the presence of LmNop56 in quiescent promastigotes, we
carry out a Western blot experiment with protein extracts
obtained from parasites harvested in early (4 days, Sta 4)
and late (7 days, Sta 7) stationary phases. As above, a
single band with a molecular mass of around 53 kDa was
observed in replicative parasites (Log; Figure 3(a)) and in

https://imagej.nih.gov/ij/

4 BioMed Research International

(a)

(b)

Figure 1: Sequence alignment and three-dimensional predicted structure of Nop56 orthologues. (a) Alignment of amino acid sequences
of Nop56 from L. major (Lm; LmjF.10.0210), T. brucei (Tb; Tb927.8.3750), T. cruzi (Tc; TCDM 07668), S. cerevisiae (Sc; YLR197W), and H.
sapiens (Hs; O00567). Identical residues in all species are indicated by black shading, while conserved residues in four organisms are denoted
by gray shading. Trypanosomatid-specific conserved residues are shaded in red. Predicted secondary structure elements in LmNop56 are
displayed on top of the linear sequence. 𝛽-strands are symbolized by arrows and 𝛼-helices by cylinders. The NOP5NT, NOSIC, and Nop
conserved domains are indicated in red, green, and blue, respectively. (b) The predicted three-dimensional modeling of Nop56 from L. major
(residues 8 to 421), T. brucei (8 to 421), and S. cerevisiae (8 to 417) was obtained with the UCSF Chimera software using the SWISS-MODEL
Template Library ID: 5wyj.3.A as a prototype. The three highly conserved domains are colored as indicated in panel (a).

nonproliferative cells (Sta 4 and 7; Figure 3(a)). The expres-
sion of LmNop56 was similar in growing and stationary
phase cells, as indicated by the loading control with 𝛼/𝛽-
tubulin (Figure 3(a)). In order to determine the subcellular
localization of LmNop56 in stationary growth phase pro-
mastigotes, indirect immunofluorescence assays were car-
ried out. Quiescent organisms were visualized as thin and
extended cells that possess an elongated nucleus and a long
flagellum (Sta 4 and Sta 7 in Figure 3(b)). LmNop56 green
signal was mainly located in the interior of the nucleolus
(Figure 3(b)). However, spherical fluorescent foci located at
the periphery of the nucleus were perceived in both early
and late stationary growth phase promastigotes (Figure 3(b);
white and black arrows). Hence, a portion of the LmNop56
protein delocalizes from the nucleolus to the nucleoplasm in
nonproliferative parasites.

3.4. Nucleolar Distribution of LmNop56 during Mitosis. To
analyze the fate of LmNop56 during closed mitosis in L.
major promastigotes, we performed double immunolabeling

of LmNop56 and 𝛼/𝛽-tubulin using a mixture of polyclonal
antibodies raised against these proteins in fixed parasites.
While the entire L. major body was illuminated by the
green fluorescence of the subpellicular microtubules array, in
interphase cells the red signal of LmNop56 is accumulated
exclusively in the nucleolus (Figure 4(a)). In contrast to
the nucleolar disassembly observed in other organisms at
the beginning of open mitosis [8, 14], our micrographs
suggest that in L. major the structure of the nucleolus is
preserved throughout the nuclear division (Figure 4(b)). At
the onset of closed mitosis, the nucleolar material (here
represented by LmNop56) spreads in the central space of the
elongated nucleus and interacts with the microtubules of the
intranuclear mitotic spindle (Figure 4(b); early mitosis). As
mitosis proceeds, LmNop56 progressively moves toward both
ends of the nucleus, probably propelled by the driving forces
of the spindle fibers and their associated motor proteins.
This hypothesis is based on the marked colocalization found
between LmNop56 and 𝛼/𝛽-tubulin (Figure 4(b); middle
mitosis). Finally, in late mitotic stages, the red fluorescent

BioMed Research International 5

Lm Tb Tc

kDa


(a)

Brightfield+MergeMergeLmNop56DNA

(b)
H4 LmNop56 LmNop56+H4 LmNop56+H4+DNA

(d)

Figure 2: Nop56 expression in trypanosomatids and subcellular localization in L. major. (a) Western blot analysis of protein extracts from
L. major promastigotes (Lm), T. brucei procyclic forms (Tb), and T. cruzi epimastigotes (Tc) was performed using an LmNop56 polyclonal
antibody. (b) Indirect immunofluorescence experiment conducted with the same anti-LmNop56 serum and an anti-mouse IgG antibody
conjugated with Alexa Fluor� 488 dye. Nuclei (N) and kinetoplast (K) in L. major promastigotes were counterstained with propidium
iodide. Nucleolar (No) localization of LmNop56 was analyzed in single optical sections obtained by confocal microscopy. (c) Double indirect
immunofluorescence assay carried out with antibodies raised against histone H4 and LmNop56. Histone H4 was revealed with anti-rabbit
IgG coupled with Alexa Fluor� 488 (green), and LmNop56 with anti-mouse IgG conjugated with Alexa Fluor� 568 (red). (d) Double
indirect immunofluorescence experiment performed with transgenic promastigotes expressing Elp3b-PTP. The antibodies employed were
anti-LmNop56 and anti-Prot C. LmNop56 (red) was detected as indicated in panel (c), whereas Elp3b was revealed with anti-rabbit IgG
coupled with Alexa Fluor� 488 (green). Images shown in panels (c) and (d) were obtained with an epifluorescence microscope; in these same
panels, DNA was stained with DAPI (blue). Size bars represent 2𝜇m.

label of LmNop56 was localized only in a particular area of the
nucleoplasm, which is poorly stained with DAPI (Figure 4(b);
late mitosis). To further analyze the nucleolar division in L.
major, we carried out double immunolabeling of Elp3b and
𝛽-tubulin. In this experiment we employed an L. major cell
line where Elp3b was labeled with a PTP tag [37], using a
mixture of antibodies that recognize the protein C epitope
and𝛽-tubulin. As shown in Figure 5, the distribution of the
Elp3b signal is very similar to that observed with LmNop56,
indicating that the nucleolus is preserved during the nuclear
division. Thus, our results strongly suggest that during
mitosis of L. major promastigotes the nucleolus persists and
appears to separate out in a relatively intact form.

3.5. Fate of T. brucei Nop56 during Nuclear Division. To
analyze the subcellular location of Nop56 in procyclic forms

of T. brucei, indirect immunofluorescence experiments were
performed using the antibody raised against LmNop56,
which recognizes the T. brucei orthologue (Figure 2(a)). The
cell bodies were stained by the green fluorescence of tubulin.
Throughout the T. brucei cell cycle, Nop56 showed a subnu-
clear distribution pattern quite similar to that described in
L. major (see Figures 2(b) and 4). During interphase, Nop56
(red) is located within a nuclear region weakly stained with
DAPI (blue) that corresponds to the nucleolus (Figure 6(a)).
In early stages of T. brucei mitosis, Nop56 was concentrated
in a still spherical nucleolus (Figure 6(b); early mitosis).
Gradually, the nucleus is extended, the mitotic spindle is
assembled, and the nucleolar material (Nop56) is dispersed
in the nucleoplasm, along the mitotic spindle (Figure 6(b);
middle mitosis). At the end of mitosis, the fluorescent signal
of Nop56 was located in the incipient nucleoli of both
resultant cells (Figure 6(b); late mitosis).

6 BioMed Research International

Log  
Sta

kDa


Tub

(a)

LmNop56+DNALmNop56+DNA LmNop56+DNA

(b)

Figure 3: Expression and subcellular distribution of LmNop56 in L. major promastigotes in stationary growth phase. (a) Westernblot analysis
of total protein extracts from promastigotes harvested in the mid logarithmic phase (Log), and early (Sta 4) and late (Sta 7) stationary
phases. The blots were probed with polyclonal LmNop56 immune serum and with 𝛼/𝛽-tubulin antibody (loading control). (b) Indirect
immunofluorescence assays performed with cells in Log, Sta 4, and Sta 7 phases using the anti-LmNop56 serum, and an anti-mouse IgG
antibody conjugated with Alexa Fluor� 488 dye (green). Nuclear and kinetoplast DNA were counterstained with DAPI (blue). Nucleolar (No)
and extra-nucleolar (arrows) green fluorescent signals were visualized with a conventional epifluorescence microscope. Size bars represent
5𝜇m.

4. Discussion

The nucleolus is a large membrane-less nuclear body where
most steps of ribosome biogenesis take place. Two impor-
tant events involved in maturation of pre-rRNA are nucle-
olytic cleavage of transcribed spacers and site-specific 2󸀠-
O-methylation of rRNA [5]. Both processes are directed by
RNA-protein complexes formed by a box C/D snoRNA and
four proteins known in human as 15.5K (Snu13p in yeast and
L7Ae in Archaea), Nop56, Nop58, and the methyltransferase
enzyme, fibrillarin (Nop1 in yeast). In Archaea, the Nop5 pro-
tein is a single homologue of eukaryotic Nop56 and Nop58
[28, 30, 31, 42]. Each box C/D snoRNP in eukaryotes contains
a single snoRNA, two copies of 15.5K and fibrillarin, and
one copy of Nop56 and Nop58 (which form a heterodimer).
In Archaea, the Nop56/Nop58 heterodimer is replaced by
a Nop5 homodimer. In silico analysis performed in the
TriTrypDB database allowed us to identify the orthologue
of Nop56 in L. major, which we characterized in this work.
Trypanosomatids also contain orthologues of 15.5K and
fibrillarin, but they do not seem to have a Nop58 orthologue.
Consequently, similarly to Archaea, it would be expected that
C/D snoRNPs in L. major and other trypanosomatids possess
Nop56 homodimers instead of the typical Nop56/Nop58
heterodimers found in eukaryotes. Future studies will help to
explore this hypothesis.

Nop56 is an evolutionarily conserved factor that orches-
trates the correct assembly and functioning of snoRNPs,

as it serves as a molecular bridge to bring together all the
core components by means of its three well characterized
modules. The assembly of box C/D snoRNPs has been
extensively studied in Archaea, where they are known as
box C/D sRNPs. The N-terminal motif, called NOP5NT,
interacts with fibrillarin to form a catalytic heterodimer
before joining, through the Nop domain, to L7Ae bound
to guide sRNA [43, 44]. In parallel, two Nop5 proteins
(each already attached to fibrillarin and L7Ae) homodimerize
via the coiled-coil region of the NOSIC motif to complete
the sRNP formation [43]. The NOP5NT, NOSIC, and Nop
domains are cataloged as preserved domains in nucleolar
proteins throughout evolution [45].

As shown by Western blot analysis, the molecular mass
of Nop56 is conserved in trypanosomatids: ∼52.7 kDa (473
aa) in L. major, ∼54.3 kDa (483 aa) in T. brucei, and ∼
53.6 kDa (481 aa) in T. cruzi (Figure 2(a)). The orthologues
in yeast (56.8 kDa, 504 aa) and human (66 kDa, 594 aa)
are larger due to an extension in the C-terminal region
(Figure 1(a)). Nevertheless, our results show that the overall
sequence of Nop56 from L. major and other trypanoso-
matids is conserved and it contains the three characteristic
Nop56 domains (NOP5NT, NOSIC, and Nop) (Figure 1(a)).
Moreover, homology modeling revealed that the hypothetical
three-dimensional structure of LmNop56 is very similar to
the model of Nop56 reported for yeast (Figure 1(b)). Thus,
the conservation of sequence and structure of LmNop56
strongly suggest that, similarly to other organisms, it may be

BioMed Research International 7

Interphase

(a)

Early
mitosis

Middle
mitosis

Late
mitosis

MergeLmNop56TubulinDNA

(b)

Figure 4: Intranuclear distribution of LmNop56 during cell division of L. major. Parasites fixed with paraformaldehyde were stained for
double indirect immunofluorescence analysis using a mixture of primary antibodies against LmNop56 and𝛼/𝛽-tubulin (to label subpellicular
microtubules and mitotic spindle) followed by anti-mouse IgG conjugated with Alexa Fluor� 568 (red) and anti-rabbit IgG coupled with
Alexa Fluor� 488 (green) secondary antibodies. DNA was stained with DAPI (blue). (a) Interphase promastigote with (bottom image) and
without (top image) brightfield. (b) L. major promastigotes at early, middle, and late stages of closed mitosis were analyzed. DNA, LmNop56,
subpellicular microtubules, and mitotic spindle (indicated with a white arrowhead) are visualized. Colocalization of DNA and proteins is
displayed in the Merge column. All images were obtained with a conventional epifluorescence microscope. K: kinetoplast; N: nucleus; No:
nucleolus. Size bar denotes 2𝜇m.

involved in the assembly and function of box C/D snRNPs
that participate in methylation [43, 44] and cleavage of the
rRNA primary transcript [28–32].

Indirect immunofluorescence assays indicated that
LmNop56 is a nucleolar protein, as the fluorescence
signal was detected at the nuclear region less stained with
propidium iodide (Figure 2(b)), histone H4 (Figure 2(c)),
and DAPI (Figure 4(a)). Colocalization analysis with the
nucleolar protein Elp3b proved that LmNop56 is located in
the nucleolus (Figure 2(d)). The main areas of overlapping
probably correspond to the fibrillar component of the
nucleolus, since Elp3b regulates transcription of rDNA in T.
brucei [41] and colocalizes with 18S rRNA genes in L. major
[37]. The exclusive location of LmNop56 in the nucleolus is
different from what has been reported in other organisms
for several nucleolar proteins, including fibrillarin, that also
localize to Cajal bodies [46]. Although Cajal bodies have not
been reported in Leishmania, electronic microscopy data
showed that the T. cruzi nucleus contains at least one Cajal
body [47, 48].

Even though a small fraction of LmNop56 was observed
outside the nucleolus in stationary phase promastigotes, most
fluorescent signal was detected within a discrete nucleolus
(Figure 3(b)). Thus, this data indicates that the nucleolus is
preserved in nonproliferative L. major cells. This is different

from T. cruzi, where the nucleolus is broken and disassembled
in stationary phase epimastigotes; consequently, in aged T.
cruzi epimastigotes, nucleolar proteins (such as Met-III and
RPA31) are dispersed throughout the nucleoplasm [49, 50]
or delocalized to the cytoplasm (fibrillarin) [51]. Changes in
nucleolar structure and scattering of nucleolar proteins have
also been observed in other organisms under nutrient starva-
tion and inhibition of rDNA transcription, conditions that are
present in stationary phase cultures. For instance, nitrogen
deprivation in S. cerevisiae causes a reduction of nucleolar
size accompanied by the nucleolar delocalization of RNA Pol
I subunits A43 and A190, which became distributed through-
out the nucleoplasm [52]. Similar results were obtained by
rapamycin, an inhibitor of protein kinase TOR (target of
rapamycin) involved in the regulation of rDNA transcription
[52]. Also, repression of RNA Pol I transcription in HeLa
cells produces segregation of the nucleoli and redistribution
of nucleolar proteins B23 and nucleolin to the cytoplasm
and the nucleoplasm, respectively [53]. The presence of
LmNop56 in the nucleolus of quiescent cells is intriguing,
considering that nonproliferative trypanosomatid cells show
a reduced level of rDNA transcription [54, 55]. It is possible
that LmNop56 remains associated with complete or partial
snRNPs that would be ready to function when favorable
growth conditions are reestablished or after differentiation

8 BioMed Research International

Early
mitosis
Middle
mitosis
Late
mitosis

MergeElp3bTubulinDNA

Figure 5: Nuclear distribution of Elp3b during the mitotic cell cycle of L. major. Promastigotes were fixed with paraformaldehyde and
stained for double indirect immunofluorescence analysis using a mixture of primary antibodies against protein C (for recombinant Elp3b-
PTP protein) and𝛽-tubulin (to label subpellicular microtubules and mitotic spindle) followed by anti-rabbit IgG coupled with Alexa Fluor�
594 (red) and anti-mouse IgG conjugated with Alexa Fluor� 488 dye (green) secondary antibodies. DNA was stained with DAPI (blue). L.
major promastigotes at early, middle, and late stages of closed mitosis were analyzed. DNA, Elp3b, subpellicular microtubules, and mitotic
spindle (indicated with a white arrowhead) are visualized. Colocalization of DNA and proteins is displayed in the Merge column. All images
were obtained with a conventional epifluorescence microscope. K: kinetoplast; N: nucleus; No: nucleolus. Size bar denotes 2𝜇m.

to infective metacyclic promastigotes. Alternatively, in aged
parasites LmNop56 might be involved in additional functions
related to cell survival or stage transition.

Little attention has been paid to the division of the nucleo-
lus at the end of mitosis in unicellular organisms. Based on the
fact that Nop56 plays significant roles as a transacting element
in ribosome biogenesis, we chose this protein as a target to
analyze the nucleolar division in L. major and T. brucei, which
undergo closed mitosis. To simplify the analysis, we divided
the mitotic process into early, middle, and late mitosis, based
on the distribution of nuclear and mitochondrial DNA,
the mitotic spindle, and LmNop56. According to previous
reports [56], early mitosis might correspond to prophase,
middle mitosis to metaphase and anaphase, and late mitosis
to telophase. When mitosis begins, Nop56 starts spreading
in the middle part of the nucleus (Figures 4 and 6). As the
cellular division advances, Nop56 is relocated to both ends of
the elongated nucleus by interacting with the mitotic spindle,
as suggested by their colocalization (Figures 4 and 6). In the
end of mitosis, two new nucleoli are clearly observed in the
still attached daughter cells. Notably, our data indicate that
the nucleolus is preserved throughout the mitotic cell division

of L. major promastigotes (Figures 4 and 5). Moreover,
they support previous results that indicated the conservation
of the nucleolus during mitosis in the insect stage of T.
brucei (Figure 6) [57]. While early studies suggested that
the nucleolus disappears when T. cruzi epimastigotes enter
mitosis [58], a recent report showed that the nucleolus does
not dissociate in the course of the cell division of this parasite
[48]. Thus, nucleolar conservation during the mitotic cycle
seems to be a distinctive feature in trypanosomatids. It would
be important to determine whether transcription of rRNA
genes remains active throughout mitosis in this group of
organisms. Since our data strongly suggest that the nucleolus
persists during the mitotic cycle of L. major promastigotes,
the presence of PNBs would not be expected. The absence of
PNBs during mitosis has been previously reported in T. cruzi
epimastigotes [48] and Giardia lamblia trophozoites [59].

The nucleolus is a dynamic NB whose main function is the
biosynthesis of ribosomes. However, this organelle appears
to be involved in other transcendental cellular processes,
including cell cycle progression and proliferation, apoptosis,
senescence, telomerase activity, and the biogenesis of several
ribonucleoprotein complexes [22, 60]. The plurifunctional

BioMed Research International 9

Interphase
(a)
Early
mitosis
Middle
mitosis
Late
mitosis

MergeTbNop56TubulinDNA

(b)

Figure 6: Subcellular localization of Nop56 during mitosis of procyclic T. brucei cells. Double Immunofluorescence assay was conducted in
cells fixed with paraformaldehyde and then stained for Nop56 and𝛼/𝛽-tubulin (to detect subpellicular microtubules and mitotic spindle),
followed by anti-rabbit IgG coupled with Alexa Fluor� 488 (green) and anti-mouse IgG conjugated with Alexa Fluor� 568 (red) secondary
antibodies. Nuclear and kinetoplast DNA were counterstained with DAPI (Blue). (a) Procyclic parasites during interphase. Size bar indicates
5𝜇m. (b) A set of representative micrographs of parasites in the different steps of closed mitosis is presented. Overlapping of DNA, tubulin,
and TbNop56 is shown in the Merge column. Intranuclear mitotic spindle is indicated by a white arrowhead. Images were obtained with a
conventional epifluorescence microscope. K: kinetoplast; N: nucleus; No: nucleolus. Size bar denotes 2𝜇m.

nucleolus hypothesis [60] was reinforced by data of pro-
teomic analysis that indicate that only∼30% of the nucleolar
protein repertoire has a role in ribosomal biogenesis [22]. As
in other eukaryotes, in L. major the most evident nucleolar
activity is the synthesis of small and large ribosomal subunits.
It remains to be determined if the nucleolus is involved in
other relevant functions in this early-diverged eukaryote.

5. Conclusions

Our results showed that Nop56 is a structurally conserved
protein found in the nucleolus throughout the cell cycle of L.
major promastigotes and procyclic T. brucei cells. Contrary
to what happens to nucleolar proteins from other eukary-
otes, we found that LmNop56 remains mainly associated
with the nucleolus in nonproliferative L. major parasites.
We also observed that during closed mitosis the nucleolar
structure, illuminated by Nop56 and Elp3b fluorescence, is
preserved and inherited to daughter nuclei as a preassembled
organelle pulled by the spindle fibers. Hence, we can speculate
that during closed mitosis the rRNA processing factors (as
LmNop56) are intimately linked to the nucleolus, probably
in the form of RNP particles. Together, the findings reported
in this manuscript significantly advance our understanding
of the basic biology of the nucleolus in trypanosomatids, a
group of early-branched eukaryotes.

Data Availability

Images from replica experiments are available from the
corresponding authors upon request.

Conflicts of Interest

The authors have no conflicts of interest to declare.

Acknowledgments

This work was supported by grant 256561 from CONACyT
to T. Nepomuceno-Mej́ıa, grant 251831 from CONACyT,
and grant IN207118 from PAPIIT (UNAM) to S. Mart́ınez-
Calvillo. We thank Dr. Miguel Tapia-Rodŕıguez for his
excellent technical assistance in confocal microscopy.

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Yang et al. J Exp Clin Cancer Res (2022) 41:25
https://doi.org/10.1186/s13046-022-02240-5

R E S E A R C H

Metabolic synthetic lethality by targeting
NOP56 and mTOR in KRAS-mutant lung cancer
Zhang Yang1†, Shun‑Qing Liang1,2†, Liang Zhao1, Haitang Yang1,3, Thomas M. Marti1, Balazs Hegedüs4,
Yanyun Gao1, Bin Zheng5, Chun Chen5, Wenxiang Wang6, Patrick Dorn1, Gregor J. Kocher1,
Ralph A. Schmid1* and Ren‑Wang Peng1*

Abstract
Background: Oncogenic KRAS mutations are prevalent in human cancers, but effective treatment of KRAS‑mutant
malignancies remains a major challenge in the clinic. Increasing evidence suggests that aberrant metabolism plays a
central role in KRAS‑driven oncogenic transformation. The aim of this study is to identify selective metabolic depend‑
ency induced by mutant KRAS and to exploit it for the treatment of the disease.

Method: We performed an integrated analysis of RNAi‑ and CRISPR‑based functional genomic datasets (n = 5) to
identify novel genes selectively required for KRAS‑mutant cancer. We further screened a customized library of chemi‑
cal inhibitors for candidates that are synthetic lethal with NOP56 depletion. Functional studies were carried out by
genetic knockdown using siRNAs and shRNAs, knockout using CRISPR/Cas9, and/or pharmacological inhibition, fol‑
lowed by cell viability and apoptotic assays. Protein expression was determined by Western blot. Metabolic ROS was
measured by flow cytometry‑based quantification.

Results: We demonstrated that nucleolar protein 5A (NOP56), a core component of small nucleolar ribonucleopro‑
tein complexes (snoRNPs) with an essential role in ribosome biogenesis, confers a metabolic dependency by regulat‑
ing ROS homeostasis in KRAS‑mutant lung cancer cells and that NOP56 depletion causes synthetic lethal susceptibility
to inhibition of mTOR. Mechanistically, cancer cells with reduced NOP56 are subjected to higher levels of ROS and rely
on mTOR signaling to balance oxidative stress and survive. We also discovered that IRE1α‑mediated unfolded protein
response (UPR) regulates this process by activating mTOR through p38 MAPK. Consequently, co‑targeting of NOP56
and mTOR profoundly enhances KRAS‑mutant tumor cell death in vitro and in vivo.

Conclusions: Our findings reveal a previously unrecognized mechanism in which NOP56 and mTOR cooperate to
play a homeostatic role in the response to oxidative stress and suggest a new rationale for the treatment of KRAS‑
mutant cancers.

Keywords: KRAS‑mutant cancer, NOP56, mTOR, ROS, Synthetic lethal vulnerability

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Background
Oncogenic mutations in the RAS family (HRAS, KRAS,
and NRAS) are the most common genetic alterations
across human cancers and occur in approximately 25% of
all tumors (COSMIC; http:// cancer. sanger. ac. uk/ cosmic).
KRAS is the predominant isoform of the RAS family pro-
teins activated by mutations (most frequently at codon
12, 13, and 61) in cancers and is responsible for 85% of all

Open Access

*Correspondence: Ralph.Schmid@insel.ch; Renwang.Peng@insel.ch
†Zhang Yang and Shun‑Qing Liang contributed equally to this work.
1 Division of General Thoracic Surgery and Department of BioMedical
Research (DBMR), Inselspital, Bern University Hospital, University of Bern,
Murtenstrasse 28, 3008 Bern, Switzerland
Full list of author information is available at the end of the article

http://orcid.org/0000-0003-1199-6520

http://creativecommons.org/licenses/by/4.0/

http://creativecommons.org/publicdomain/zero/1.0/

http://cancer.sanger.ac.uk/cosmic

http://crossmark.crossref.org/dialog/?doi=10.1186/s13046-022-02240-5&domain=pdf

Page 2 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

RAS-driven cancers, particularly pancreatic, colon, and
non-small cell lung cancer (NSCLC) [1]. Mutant KRAS is
associated with poor prognosis and treatment resistance.
However, unlike NSCLC with less frequent oncogenic
drivers (e.g., EGFR, ALK, MET1, and ROS1) that respond
significantly to selective kinase inhibitors [2], effec-
tive therapies specifically targeting KRAS-mutant can-
cers remains a challenge [2, 3]. Despite recent progress
of immune checkpoint inhibitors of programmed death
1 (PD1) and the ligand PD-L1 in treating NSCLC, they
fail to discriminate KRAS-mutant from other NSCLC [4].
Covalent KRAS inhibitors have demonstrated promise in
preclinical models, but they are only effective for a spe-
cific KRAS-G12C mutant allele and additional agents are
needed to optimize the anticancer efficacy [5–7]. Target-
ing KRAS downstream effectors, such as the mitogen-
activated protein kinase (MAPK) RAF/MEK/ERK, has
been widely pursued, but the pleiotropic nature and com-
plex interplay among individual signaling cascades and
toxicity ensuing from sustained inhibition of multiple
KRAS effector pathways has hindered the translational
potential of the strategy [8, 9]. Consequently, identifica-
tion of new targets for innovative treatment strategies
tailored to KRAS-mutant cancers still represents a press-
ing need [3].

The concept to target KRAS synthetic lethality, prem-
ised by the notion that oncogenic KRAS signaling fuels a
unique cell state, manifested by adaptation to oncogenic
stress and transcriptional, translational and metabolic
reprogramming, and that interfering with this KRAS-
driven cell state may result in selective cytotoxicity for
KRAS-mutant cancer, provides an alternative strategy for
treating KRAS-driven cancers [10, 11]. Indeed, exploit-
ing cancer cell vulnerabilities contextually induced by
mutant KRAS, in particularly the mechanisms critical
for surveillance of oncogene-dependent cellular stresses
(genotoxic, proteotoxic, and metabolic) that are permis-
sive for strong oncogenic signaling, has not only provided
promising therapeutic avenues but also a wealth of infor-
mation on the fundamental principles of KRAS-induced
tumorigenicity [12–14]. Activating KRAS mutations
deregulate mitosis, nuclear export, redox, and mitochon-
drial activity, and KRAS-mutant cancer cells have con-
sequently been shown to have a greater dependency on
the functions of non-oncogenes [e.g., PLK1, XPO1, and
MRPL52 (a component of the mitochondrial large ribo-
somal subunit)] that play critical roles in their respective
processes [12–16], suggesting that targeting non-onco-
gene addiction is an attractive approach for the treatment
of KRAS-mutant cancer [17, 18].

NOP56 (nucleolar protein 5A or NOL5A) is a rib-
onuclear protein, which, together with fibrillarin
(FBL), NOP58 (nucleolar protein 58), and nonhistone

chromosome protein 2-like 1 (NHP2L1 or SNU13p,
15.5 kDa), forms the core protein set of box C/D small
nucleolar ribonucleoprotein complexes (snoRNPs) that
play an essential role in ribosome assembly by methylat-
ing rRNA at the 2′-O-ribose and modulating ribosomal
RNA (rRNA) processing [19, 20]. Recent evidence sug-
gests that NOP56 and the other snoRNPs are the novel
group of nucleolar proteins that promote cell transfor-
mation and tumorigenesis [21, 22]. Indeed, ribosome
biogenesis is the only cellular process in which a large
number of genes harbor evolutionarily conserved MYC-
binding sites [21]. NOP56 is overexpressed in Burkitt’s
lymphoma and other cancers and serves as a marker of
poor prognosis [23]. In particular, NOP56 is required for
MYC-induced cell transformation and tumor growth in
Burkitt’s lymphoma [21]. NOP56 may also have extra-
ribosomal functions that remain to be discovered.
Nevertheless, the activity of snoRNPs in oncogenic trans-
formation suggests that they are promising therapeutic
targets for cancer treatment [24, 25].

In this study, we reported an unexpected function of
NOP56 in metabolic stress response and a previously
unrecognized metabolic synthetic lethality by target-
ing NOP56 and mTOR in KRAS-mutant cancers. Based
on integrated analyses of RNAi- and CRISPR-mediated
functional genomics [12, 16, 26], we identified NOP56
as a novel metabolic dependency of KRAS-mutant can-
cer by regulating homeostasis of reactive oxygen spe-
cies (ROS) that plays a well-established role in mutant
KRAS-induced tumorigenesis [27–30]. Depletion of
NOP56 impairs the response to oxidative stress, which
renders KRAS-mutant cancer cells highly dependent on
mTOR signaling for survival and particularly vulnerable
to mTOR inhibition. Consequently, co-targeting NOP56
and mTOR enhances apoptotic death of KRAS-mutant
lung cancer cells in vitro and in vivo. We further delin-
eated that mTOR activation upon NOP56 depletion is
mediated by IRE1α-mediated unfolded protein response
(UPR). These results uncover a previously unknown
mechanism by which NOP56 cooperates with UPR and
mTOR to regulate metabolic stress and a novel syn-
thetic lethal strategy for the treatment of KRAS-mutant
cancers.

Materials and methods
Cell culture and reagents
Cancer cell lines used in this study (Table S1) were
obtained from American Type Culture Collection
(ATCC, Manassas, VA, USA). Cells were cultured
in RPMI-1640 medium or Medium 199 (Cat. #8758
and #4540; Sigma-Aldrich, St. Louis, MO, USA) sup-
plemented with 10% fetal bovine serum/FBS (Cat.
#10270–106; Life Technologies, Grand Island, NY, USA)

Page 3 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

and 1% penicillin/streptomycin solution (Cat. #P0781,
Sigma-Aldrich). The cells were authenticated by DNA
fingerprinting and confirmed free from mycoplasma con-
tamination (Microsynth, Bern, Switzerland). All inhibi-
tors used in this study were listed in Table S2.

PF139 and PF563 lung cancer cells were established
from lung adenocarcinoma malignant pleural effusion
and pleural carcinosis specimens of a 67 year-old female
patient and a 75 year-old male patient, respectively, at the
time of diagnosis prior to any treatment [31]. Authenti-
cation was performed by SNP based cell identification
(Multiplexion, Heidelberg, Germany).

Cell viability and clonogenic survival assay
Lung cancer cells seeded in 96-well plates (2500 cells/
well) were dosed 24 h later with different inhibitors for
72 h. Cell viability was determined by PrestoBlue (PB)
Cell Viability Reagent (ThermoFisher Scientific) by fol-
lowing the manufacturer’s instructions [14, 31]. The PB
reagent was added into media directly (1:10 dilution)
and incubated for 30 min-2 h and then the fluorescence
was read (excitation 570 nm; emission 600 nm) at recom-
mended time of incubation. The efficacy of drugs on cell
growth was normalized to untreated control. Each data
point was generated in triplicate and each experiment
was done three times (n = 3). Best-fit curve was generated
in GraphPad Prism [(log (inhibitor) vs response (−vari-
able slope four parameters)]. Error bars are mean ± SD.
The combination index (CI) was calculated by ComboSyn
software (ComboSyn Inc., http:// www. combo syn. com/).

Clonogenic assay was done as we described previously
[14, 31–33]. In brief, cells seeded in 6-well plates (3000
cells/well) were dosed 24 h later and continually treated
with rapamycin for 7 days (refresh drugs every 3 days),
the resulting colonies were stained with crystal violet
(0.5% dissolved in 25% methanol).

Apoptosis assays
Lung cancer cells were treated for 72 h with vehicle or
rapamycin. After treatment, cells in the supernatant and
adherent to plates were collected, washed with PBS and
pooled before suspended in binding buffer and stained
with the Annexin V Apoptosis Detection Kit -FITC (Cat.
#88–8005; Thermo Fisher Scientific, Waltham, MA,
USA) according to the manufacturer’s instructions. Flow
cytometry analysis was performed on a BD Biosciences
LSRII flow cytometer.

Gene silencing by small interfering (siRNA), short hairpin
RNAs (shRNA) and single‑guide RNAs (sgRNA)
Transient knockdowns were mediated by siRNAs.
Cells cultured in triplicate at 50–70% confluency
were transfected using SiTran1.0 (TT300001; Origene

Technologies, Rockville, MD, USA) according to the
manufacturer’s protocol. NOP56 (CAT#: SR307156),
EIF4E (CAT#: SR320018), RPS6 (CAT#: SR304160), RAP-
TOR (CAT#: SR324724), and RICTOR (CAT#: SR326062)
were knocked down by specific pooled siRNA duplexes
purchased from OriGene Technologies, with control
siRNA Duplex as a negative control.

Stable knockdown of NOP56 was achieved via len-
tiviral delivery of NOP56 Human shRNA Plasmid Kit
(SHCLND_006392, MERCK). A scramble shRNA was
used as a control. Lentiviral particles were generated
and cells infected according to the protocol from Broad
Institute. The supernatant containing lentiviruses was
collected, filtered through 0.45 μM filters, and stored in
aliquots at − 80 °C, or immediately used to infect recipi-
ent cells. After infection, cells were selected in puromycin
(1.5 μg/ml) and further passaged in culture for functional
assays. NOP56 knockout was performed via a CRISPR/
Cas9 and non-homology mediated approach using the
NOL5A (NOP56) Human Gene Knockout Kit (CAT#:
KN411153; OriGene Technologies) according to the
manufacturer’s protocol.

Quantitative real‑time PCR (qRT‑PCR)
Total RNA was isolated and purified using RNeasy Mini
Kit (Qiagen, Hilden, Germany). Complementary DNA
was synthesized by the High capacity cDNA reverse tran-
scription kit (Applied Biosystems, Foster City, CA, USA)
according to manufacturer’s instructions. Real time PCR
was performed in triplicate on a 7500 Fast RealTime PCR
System (Applied Biosystems) using TaqMan primer/
probes (Applied Biosystems): HSPA5, Hs00607129_gH;
ERN1, Hs00980095_m1; EIF2AK3, Hs00984003_m1;
ATF4, Hs00909569_g1; DDIT3, Hs00358796_g1, with
GAPDH (Hs02786624_g1) and ACTB (Hs01060665_g1)
used as endogenous normalization controls.

Immunoblotting, immunohistochemistry
and immunofluorescence
Cell lysates were prepared and western blot analysis was
performed as described [14, 31]. In brief, equal amounts
of protein lysates resolved by SDS-PAGE (Cat. #4561033;
Bio-Rad Laboratories, Hercules, CA, USA) and trans-
ferred onto nitrocellulose membranes (Cat. #170–4158;
Bio-Rad). Membranes were then blocked with blocking
buffer (Cat. #927–4000; Li-COR Biosciences, Bad Hom-
burg, Germany) for 1 h at room temperature (RT) and
incubated with appropriate primary antibodies over-
night at 4 °C (Table S3). IRDye 680LT-conjugated goat
anti-mouse IgG (Cat. #926–68,020) and IRDye 800CW-
conjugated goat anti-rabbit IgG (Cat. #926–32,211)
from Li-COR Biosciences were used at 1:5000 dilutions.
Finally, signals of membrane-bound secondary antibodies

http://www.combosyn.com/

Page 4 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

were imaged using the Odyssey Infrared Imaging System
(Li-COR Biosciences).

For immunofluorescence, tumor cells grown on poly-
lysine-treated coverslides were fixed with 4% paraform-
aldehyde for 15 min at RT and permeabilized with cold
methanol (− 20 °C) for 5 min or with 0.1% Triton X-100/
PBS at RT for 15 min before incubated overnight at 4 °C
with primary antibodies (Table S3). The cells were incu-
bated for 1 h at RT with Alexa Fluor 647 goat anti-mouse
IgG (Cat. #A21236) or Alexa Fluor 488 goat anti-Rabbit
IgG (Cat. #A11034) from Invitrogen (Eugene, OR, USA).
Nuclei were counterstained by 4′,6-diamidino-2-phe-
nylindole. Images were acquired on a ZEISS Axioplan 2
imaging microscope (Carl Zeiss MicroImaging, Göttin-
gen, Germany) and processed using Adobe Photoshop
CS6 v.13 (Adobe Systems, San Jose, CA, USA).

Immunohistochemical study was performed as we
described previously [31, 32]. In brief, surgically removed
xenograft tumors were formalin-fixed and paraffin-
embedded (FFPE). FFPE tumors were sectioned at 4 μm,
deparaffinized, rehydrated and subsequently stained
with hematoxylin and eosin (H&E) and appropriate anti-
bodies (Table S3) using the automated system BOND
RX (Leica Biosystems, Newcastle, UK). Visualization
was performed using the Bond Polymer Refine Detec-
tion kit (Leica Biosystems) as instructed by the manu-
facturer. Images were acquired using PANNORAMIC®
whole slide scanners, processed using Case Viewer
(3DHISTECH Ltd.). The staining intensities of the whole
slide (two tumors/group) were quantified by QuPath
software.

In vivo mouse study
Mouse studies were conducted in accordance with Insti-
tutional Animal Care and Ethical Committee-approved
animal guidelines and protocols. All mouse experi-
ments were performed in age- and gender-matched
NSG (NOD-scid IL2Rγnull) as we previously described
[31, 32]. Tumor cells in DMEM (H460-shScrambled or
H460-shNOP56) 1:1 mixed with BD Matrigel Basement
Membrane Matrix (Cat. #356231; Corning, NY, USA)

were subcutaneously inoculated in left and right flanks
(0.5 × 106/injection). When tumors were palpable, mice
were randomly assigned to treatment groups: 1) control;
2) rapamycin (0.1 mg/kg, i.p, 5 days/week) for 5 weeks.
Tumors were measured every 3 days, with their size cal-
culated as follows: (length × width2)/2. For survival anal-
ysis, the mice were closely monitored on a daily basis,
and the size of tumors was measured with a caliper every
4–5 days. Mice were sacrificed when the tumor volume
reached 1500 mm3.

Public databases
To identify synthetic lethal targets in KRAS-mutant can-
cers, we interrogated functional genomics dataset of
CRISPR/Cas9 knockout and RNAi/shRNA knockdown
screens from published studies: whole genome RNAi
screens in DLD-1 colon cancer cells [12] and in KRAS-
mutant lung cancer cells (H2122, H2009, HCC44, H460,
H1155) [16], genome-wide CRISPR/Cas9 loss of function
screens in KRAS-mutant leukemia cells (PL-21, SKM-
1, NB4) [26]. To minimize the effects of cancer lineage
and histological subtype, we selected DLD-1 (colon),
H460 (large cell lung carcinoma), H2122 (lung adeno-
carcinoma), SKM-1 (without PML-RARA fusion) and
NB4 (PML-RARA ) for further analysis, which identified
a number of common candidates (n = 21) as KRAS syn-
thetic lethal partners (Fig. 1).

Interrogation of publicly available datasets was per-
formed as we have described [14, 31]. Specifically, tran-
scriptomic data of lung, pancreatic and colon cancer
were obtained from the Cancer Genome Atlas (TCGA)
(https:// portal. gdc. cancer. gov/ proje cts/ TCGA). Gene
set enrichment analysis (GSEA) was performed by using
GSEA software. The transcriptomic dataset (GSE15212)
used for GSEA was derived from KRAS-mutant colon
cancer cell line (SW480) treated with NOP56-specific
siRNAs and downloaded from the Gene Expression
Omnibus (GEO) database. For survival analysis, tran-
scriptomic gene expression and corresponding survival
data were extracted and analyzed by using the “max-
stat”, “survival”, and “survminer” packages in R software

(See figure on next page.)

Fig. 1 NOP56 confers a metabolic dependency in KRAS‑mutant cancers. A, Venn diagram showing common essential genes in KRAS‑mutant
cancer cells. Data are based on the published studies, with the 21 common genes listed on the right. B, Network analysis of the 21 common genes
by STRING. C, NOP56 mRNA expression in KRAS‑mutant lung cancer (LC), pancreatic cancer (PC) and colon cancer (CC) versus KRAS‑wild‑type
(WT ) cancers in patient samples from TCGA. D, Prognostic values of NOP56 expression across TCGA lung adenocarcinoma (left), pancreatic cancer
(middle) and colon cancer (right) cohorts harboring KRAS mutations. Kaplan–Meier survival analyses were stratified by the optimal cut‑off value of
NOP56 mRNA levels. E, Gene set enrichment analysis (GSEA) revealed significant enrichment of oxidative phosphorylation and ROS pathway gene
signatures in NOP56‑depleted KRAS‑mutant cancer cells (SW480). The GEO dataset GSE15212 was used for GSEA. F, H358 and H460 cells transfected
with NOP56-specific or control siRNAs were treated (72 h post‑transfection) with 300 μM H2O2 for 6 h, followed by incubation with H2DCFDA
for 30 min, and analyzed by flow cytometry. Quantification of relative ROS levels was shown in the right. Data are shown as the mean ± SD
(n = 3). *P < 0.05,***P < 0.001, ****P < 0.0001 by two‑way ANOVA with Tukey’s multiple comparisons test. G, H358 and H460 cells transfected with NOP56‑specific or control siRNAs were subsequently (72 h post transfection) treated with vehicle (DMSO) or 300 μM H2O2 for 6 h before apoptotic assay. Data were shown as mean ± SD (n = 3). *P < 0.05 and ***P < 0.001 by two‑way ANOVA with Tukey’s multiple comparisons test

https://portal.gdc.cancer.gov/projects/TCGA

Page 5 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

Fig. 1 (See legend on previous page.)

Page 6 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

(version 3.6.0). Patients were divided into two groups
(high_ NOP56 versus low_ NOP56) based on the optimal
cutoff value of NOP56 transcripts across all patients to
plot the Kaplan–Meier survival curves.

For correlative analysis of NOP56 expression with sen-
sitivity (IC50) to mTOR inhibitors, gene expression data
and drug response profiles were downloaded from Can-
cer Cell Line Encyclopedia (CCLE) and Genomics of
Drug Sensitivity in Cancer (GDSC) databases, respec-
tively. Correlation analysis was performed using R soft-
ware (version 3.6.0).

Statistical analysis
Statistical analyses were performed using GraphPad
Prism 7.01 (GraphPad Software Inc., San Diego, CA,
USA) unless otherwise indicated. In all studies, data rep-
resent biological replicates (n) and are depicted as mean
values ± SD or mean values ± SEM as indicated in the
figure legends. In all analyses, P values less than 0.05 were
considered statistically significant. For the survival anal-
ysis, patients were grouped by gene expression, where
‘high’ and ‘low’ expression groups were stratified by the
optimal cut-off value.

Results
NOP56 confers a metabolic dependency by regulating ROS
homeostasis in KRAS‑mutant lung cancer
To identify therapeutic vulnerabilities in KRAS-mutant
cancers, we performed integrated analysis of shRNA- and
CRISPR-based functional genomics of previously pub-
lished studies [12, 16, 26]. To minimize lineage-specific
effects, we analyzed whole-genome dropout screen data-
set in KRAS-mutant lung (H460, H2122), colon (DLD-1),
acute promyelocytic leukemia (NB4) and acute myeloid
leukemia (SKM-1) cancer cells, which identified 21 com-
mon genes whose loss of function is synthetic lethal with
mutant KRAS alleles in distinct cancer lineages (Fig. 1A;
Table S4). The protein products of these genes fall into
several functional categories, with FBL, NOP56, PLK1
and XPO1 as a core set based on their interaction net-
work (Fig. 1B). Remarkably, PLK1 and XPO1 have been
reported to be selectively required for KRAS-mutant
cancers by counteracting mitotic and nuclear export
stress associated with KRAS-induced tumorigenesis [12,
16], and our recent study has implicated PLK1 in meta-
bolic stress response of KRAS-mutant cancers [14]. FBL
has also been assigned as a promising target in cancers
[34, 35], suggesting the power of functional genom-
ics in identifying oncogene-specific vulnerabilities and
the accountability of our analyses. In the present study,
we investigated the function of NOP56 in KRAS-mutant
cancers.

Our investigations began with NOP56 knockdown
using small interfering RNAs (siRNAs), which revealed
that downregulation of NOP56 significantly inhibited
the proliferation of numerous KRAS-mutant lung (H358,
H460, A549, PF563, PF139), pancreatic (MIAPaCa,
HPAF-II) and colon (HCT-116, DLD-1) cancer cells,
which differ not only in tumor lineages and histological
subtypes but also in KRAS mutations, e.g., G12C, G12D,
Q61H, etc. (Fig. S1A, B; Table S1). Notably, NOP56
silencing also inhibited NRAS-mutant lung cancer H1299
cells, although the effects on EGFR-mutant (EBC-1) or
FGFR1-amplified (H520) lung cancer cells were neg-
ligible (Fig. S1A, B). Supporting these observations,
KRAS-mutant lung, pancreatic and colon cancer showed
significantly higher expression of NOP56 than KRAS-WT
tumors (Fig. 1C) and patients with KRAS-mutant lung
adenocarcinoma, pancreatic and colon cancer charac-
terized by a higher NOP56 level are associated with sig-
nificantly shorter survival (Fig. 1D). In contrast, NOP56
expression is not a prognostic marker for KRAS-mutant
lung, pancreatic and colon cancers (Fig. S1C). These
results indicate a unique function for NOP56 in KRAS-
mutant cancers.

To explore NOP56 functions in KRAS-mutant malig-
nancies, we profiled the transcriptomic gene expres-
sion data of a previous study [36], whereby NOP56 in
KRAS-mutant colon cancer cells (SW480) was silenced
by siRNAs. Our analysis revealed that high expression
of NOP56 was positively correlated with the gene signa-
ture of KRAS signaling (Fig. S1D), in line with the above
results (Fig. 1A-D; Fig. S1A, B), and that, importantly,
siRNA-mediated NOP56 knockdown led to significant
enrichment of the gene sets involved in ROS pathway
(consisting of 49 genes upregulated by ROS) and oxida-
tive phosphorylation (a set of 200 genes encoding pro-
teins involved in oxidative phosphorylation), the latter
representing a major source of ROS production (Fig. 1E),
suggesting a possible role for NOP56 in the suppres-
sion of metabolic ROS that is critical for KRAS-induced
tumorigenesis [27–30]. Supporting this notion, NOP56
knockdown (KD) by siRNAs significantly upregulated
ROS in H358 and H460 cells, and H2O2 treatment, which
elevated the already high level of oxidative stress, pro-
voked significantly greater apoptosis in NOP56 KD H358
and H460 cells than the control counterparts (Fig. 1F, G).
These results uncover NOP56 as a metabolic dependency
in KRAS-mutant cancer by exerting a previously unrec-
ognized role in the surveillance of oxidative stress.

NOP56 suppression evokes IRE1α‑mediated UPR
to mitigate oxidative stress
Next, we investigated the mechanism that KRAS-mutant
cancer cells utilize to orchestrate cytotoxic ROS upon

Page 7 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

NOP56 depletion. GSEA of transcriptomic dataset [36]
revealed that NOP56 knockdown significantly enriched
the genes involved in the unfolded protein response/UPR
(a set of 113 genes upregulated during UPR) in KRAS-
mutant cancer cells (Fig. 2A), suggesting that tumor
cells might engage the UPR to protect from NOP56 KD-
induced surge of cytotoxic ROS. To test this possibility,
we knocked down NOP56 in KRAS-mutant lung cancer
cells (H358, H460) by using short-hairpin RNAs (shRNA)
(Fig. S2A, B). In contrast to the results from siRNA-
mediated acute depletion, stable expression of two inde-
pendent shRNAs showed negligible effects on H358 and
H460 proliferation (Fig. S2C, D), which may be due to
the activation of compensatory mechanisms. Impor-
tantly, several UPR genes, in particular ERN1 and HSPA5
encoding the ER stress sensor IRE1α and the chaperon
protein BiP, respectively, were markedly upregulated in
NOP56-depleted H358 and H460 cells (Fig. 2B). West-
ern blot confirmed the increase of BiP, IRE1α, XBP-1 s
(IRE1α effector) and of the master UPR transcription fac-
tor HSF-1 (heat shock factor 1) and PDI (protein disulfide
isomerase), an important ER chaperone induced during
ER stress by carrying out a redox reaction and respon-
sible for the formation of disulfide bonds in proteins
(Fig. 2C). Notably, p38 MAPK, a key stress-responsive
kinase and an UPR effector [37], was highly phosphoryl-
ated (activated) in NOP56-depleted H358 cells (Fig. 2C).
Moreover, IRE1α KD blunted p-p38, p-AKT (T308),
p-MNK1, p-eIF4E and p-S6 in NOP56-depleted H358
cells, indicating that IRE1α-mediated UPR acts upstream
of p38 signaling (Fig. 2D).

Importantly, genetic (siRNA) and pharmacological
(4μ8C, an inhibitor of IRE1α) inhibition of IRE1α pref-
erentially impaired NOP56 KD H358 and H460 cells,
manifested by significantly greater proliferative inhibition

and apoptotic induction in these cells than in control
cells (Fig. 2E, F; Fig. S2E, F). Importantly, the increase of
IRE1α KD-induced apoptotic cell death was paralleled by
ROS upregulation, and addition of NAC, an ROS scav-
enger largely dampened IRE1α KD-induced apoptosis
(Fig. 2F, G), supporting a role for the UPR in response to
oxidative stress. Similarly, genetic and pharmacological
inhibition (with KRIBB11) of HSF1 suppressed the pro-
liferation and evoked apoptotic cell death to a markedly
greater extent in NOP56 KD H358 cells than in control
cells (Fig. 2H-J).

The outcome of UPR ranges from adaptation to apop-
tosis [38] and, as such, perturbations of ER homeostasis
in cells with an already high level of ER stress, e.g., treat-
ment with bortezomib and tunicamycin that induce per-
sistent ER stress by targeting the 26S proteasome and the
ER chaperone BiP, respectively, evoke programmed cell
death [38, 39]. Indeed, NOP56 KD H358 and H460 cells
with high basal levels of ROS are highly susceptible to
bortezomib and Tunicamycin compared to control cells
(Fig. 2K, L).

Thus, targeting NOP56 disrupts ROS homeostasis and
induces IRE1α-mediated UPR in KRAS-mutant lung can-
cer cells.

IRE1α‑mediated UPR fuels mTOR signaling via p38 MAPK
To identify cellular processes that may present thera-
peutic vulnerabilities in NOP56 KD cells, we performed
synthetic lethal chemical screens with small-molecule
drugs (n = 22) that interrogate various oncogenic path-
ways, with the ER stress inducers (bortezomib and
HA15) included as positive controls (Table S2). Our
screens showed that, except for bortezomib and HA15,
LY294002, AZD5363, and rapamycin, inhibitors of the
PI3K/AKT/mTOR pathway, preferentially suppressed

Fig. 2 NOP56 depletion evokes IRE1α‑mediated UPR. A, NOP56 depletion led to significant enrichment of the UPR gene signature in KRAS‑mutant
cancer cells. GSEA was based on the GEO dataset GSE15212. B, Transcriptional quantification (qRT‑PCR) of UPR genes in H358 and H460 cells
expressing control (sh Scram) or NOP56‑specific shRNA (sh NOP56a). C, Immunoblots of H358 and H460 cells expressing scrambled control or
NOP56‑specific shRNAs. D, H358 cells expressing scrambled control or NOP56‑specific shRNAs were transfected with IRE1α-specific or control siRNAs
for 72 h before immunoblotting. E, Clonogenic assay of H460 and H358 cells expressing scrambled control or NOP56‑specific shRNAs after treated
with indicated doses of 4μ8C (IRE1α inhibitor). Representative images are shown. F, H358 cells expressing scrambled control or NOP56‑specific
shRNAs were transfected with IRE1α-specific or control siRNAs for 72 h, in the presence or absence of NAC (2.5 mM) before apoptosis assay. Data are
presented as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 and ns P>0.05 by two‑way ANOVA with Tukey’s multiple comparisons test. G, H358 cells
expressing scrambled control or NOP56‑specific shRNAs were transfected with IRE1α-specific or control siRNAs for 72 h, in the presence or absence
of NAC (2.5 mM). Cells were then washed, incubated with H2DCFDA for 30 min, and analyzed by flow cytometry. Quantification of relative ROS
levels was shown in the right. Data are presented as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 and ns P>0.05 by two‑way ANOVA with Tukey’s
multiple comparisons test. H, H358 cells expressing scrambled control or NOP56‑specific shRNAs were transfected with HSF1-specific or control
siRNAs for 72 h before apoptotic assay. I, H358 cells expressing scrambled control or NOP56-specific shRNAs were transfected with HSF1-specific or
control siRNAs for 72 h before immunoblot analysis. Data are presented as mean ± SD (n = 3). ***P < 0.001, ****P < 0.0001 and ns P>0.05 by two‑way
ANOVA with Tukey’s multiple comparisons test. J, Clonogenic assay of H460 and H358 cells expressing scrambled control or NOP56‑specific shRNAs
after treated with the indicated doses of KRIBB11 (HSF1 inhibitor). Representative images are shown. K, L, Clonogenic assay of H460 and H358
cells expressing scrambled control or NOP56‑specific shRNAs after treated with the indicated doses of the ER stress inducer bortezomib (K) or
tunicamycin (L). Representative images are shown

(See figure on next page.)

Page 8 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

Fig. 2 (See legend on previous page.)

Page 9 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

the viability of NOP56 KD cells, gauged by their IC50
decrease in NOP56 KD H358 and H460 cells versus con-
trol cells (Fig. 3A; Fig. S3A). The greatest change in sen-
sitivity was conferred by rapamycin, which was 0.7 μM
and 1.0 μM in H358_shNOP56a and H358_shNOP56b
but 12.0 μM in H358_Scr cells, with a selectivity index
(IC50 in control cells / IC50 in NOP56 KD cells) of 17- and
12-fold, respectively (Fig. 3A; Fig. S3A). These observa-
tions were validated by independent assays, in which
NOP56 KD sensitized H358 and H460 cells to PI3K/
AKT inhibitors (LY294002, AZD5363), anti-mTOR drugs
(rapamycin, everolimus), and ER stress inducers (bort-
ezomib and HA15) (Fig. 3B,C; Fig. S3B,C). Importantly,
CRISPR/Cas9-mediated knockout of NOP56 dramati-
cally increased the sensitivity of KRAS-mutant (H358,
H460) but not of wild-type (H520, H1703) lung cancer
cells to rapamycin (Fig. S3D, E).

Moreover, examining gene expression data of KRAS-
mutant cancer cells [36] revealed that NOP56 silencing
significantly enriched the mTOR gene signature (Fig. 3D).
Mining TCGA and Genomics of Drug Sensitivity in Can-
cer (GDSC) databases showed that NOP56 expression is
negatively correlated with that of mTOR pathway genes
in patients with KRAS-mutant lung adenocarcinomas
(Fig. 3E) and that NOP56 mRNA levels are a predictive
marker of sensitivity (IC50) to rapamycin in KRAS-mutant
cancer cell lines but not in KRAS-wild-type cancer cells
(Fig. 3F). These data support our in vitro results (Fig. 3A-
C; Fig. S3A-C) and further suggest a reciprocal interplay
between NOP56 and mTOR signaling.

Indeed, siRNA-mediated NOP56 KD, which slightly
increased ribonucleolar proteins (e.g., NOP58, FBL),
markedly induced AKT/mTOR (p-AKT, p-mTOR, pS6),
translation initiation (p-eIF4E) and the stress-responsive
p38 MAPK in H358 cells in a time-dependent man-
ner (Fig. 3G), as did shRNA-mediated stable NOP56

KD in H358 and H460 cells, but not in KRAS-WT lung
cancer H1703 cells (Fig. 3H; Fig. S3G). Importantly,
NOP56 KD sensitized KRAS-mutant lung (A549), colon
(HCT-116, DLD-1, and LS174T), pancreatic (MIAPaCa,
HPAF-II) and primary KRAS-mutant lung cancer cells
(PF563, PF139) to rapamycin, as well as NRAS-mutant
lung cancer H1299 cells but not KRAS-wild-type H2405
(BRAF-mutant), EBC-1 (EGFR-mutant), H1993 (MET
amplification) and H520 (FGFR1 amplification) cells (Fig.
S3H, I). These results reinforce the notion that NOP56
plays a unique role in KRAS-mutant cancer.

Our results demonstrated that NOP56 and the IRE1α-
mediated UPR act upstream of p38 and mTOR signal-
ing (Fig. 2C, D; Fig. 3G), suggesting a signaling cascade
from the UPR to mTOR via p38 MAPK. To confirm this,
we targeted p38 with the specific inhibitor SB203580,
which, as expected, barely affected the upstream IRE1α-
dependent UPR (p-IRE1α, XBP-1 s), but strikingly damp-
ened the MNK-eIF4E axis and mTOR signaling (p-AKT,
p-S6) in H358 cells (Fig. 3I). Importantly, SB203580
exposure not only decreased activity of the mTOR path-
way but also increased PARP expression and promoted
PARP cleavage (Fig. 3I), concurrent with substantially
elevated cytotoxicity on NOP56 KD H460 and H358
cells compared to that on control cells (Fig. 3J). Together,
these results unravel a signaling cascade from the IRE1α-
mediated UPR to p38 MAPK and to mTOR signaling in
KRAS-mutant lung cancer upon NOP56 suppression.

Synthetic lethality by targeting NOP56 and mTOR
in KRAS‑mutant lung cancer
Our findings that NOP56 KD cells are exposed to higher
levels of metabolic ROS and display a greater dependency
on UPR-activated mTOR signaling suggest a synthetic
lethal vulnerability in KRAS-mutant cancer (Figs. 1, 2,
3). To test this hypothesis, we treated NOP56 KD H358

(See figure on next page.)
Fig. 3 IRE1α‑mediated UPR fuels mTOR signaling via p38 MAPK. A, H358 cells expressing scrambled control or NOP56‑specific shRNAs were treated
with different inhibitors, with bar graphs illustrating sensitivity increase after NOP56 KD. Fold changes of IC50 values were presented as IC50 of
rapamycin in H358 cells expressing scrambled shRNA (sh_Scrambled) compared to that in H358 cells expressing NOP56‑targeted shRNAs (shNOP56
a/b). Data presented as mean (n = 2). B, Clonogenic assay of H358 and H460 cells expressing control shRNA (sh_Scr) or NOP56‑specific shRNAs
(sh‑a, sh‑b) after treatment with indicated doses of rapamycin or everolimus. Representative images are shown. The heatmap (right) indicates
the percentage of viable cells after the treatment, based on quantification of clonogenic results (left). Data are presented as mean ± SD (n = 3). C,
Growth inhibition of H358 and H460 cells expressing NOP56‑specific shRNAs (sh NOP56a, sh NOP56b) or control shRNAs (sh Scram) after treated for
72 h with the mTOR inhibitors (rapamycin, everolimus). Data are presented as mean ± SD (n = 3). D, NOP56 silencing significantly enriched the mTOR
gene signature in KRAS‑mutant cancer cells. GSEA was performed based on the GEO dataset GSE15212. E, Negative correlation of NOP56 mRNA
levels with mTOR gene signature (mTOR pathway score) as determined in a TCGA cohort of patients with KRAS‑mutant lung adenocarcinoma.
Pearson and Spearman coefficient, as well as the significance (p‑value), were determined using R software (Cor.test function). F, NOP56 expression
is a predictive marker of sensitivity (IC50) to rapamycin in KRAS‑mutant cancer cell lines (n = 18) but not in KRAS‑wide‑type cancer cell lines (n = 82).
Drug response profiles were downloaded from the GDSC (Genomics of Drug Sensitivity in Cancer) database. G, Immunoblots of H358 cells after
transfection with scramble control siRNAs (si‑Control) for 72 h (−) or NOP56‑specific siRNAs (si‑NOP56) for different time points (24 h, 48 h, 72 h and
96 h). H, Immunoblots of H358 and H460 cells expressing scrambled control or NOP56‑specific shRNAs. I, Immunoblots of H358 cells expressing
scramble control or the NOP56‑specific shRNAs after treated with the p38 inhibitor SB203580 (5 μM) for 24 h. J, Clonogenic assay of H460 and H358
cells expressing control or NOP56‑specific shRNAs after treated with the p38 inhibitor SB203580. Representative images are shown

Page 10 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

Fig. 3 (See legend on previous page.)

Page 11 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

and control cells with rapamycin, which, as expected,
decreased mTOR effectors (e.g., p-S6, p-eIF4E) (Fig. 4A)
that were the otherwise adaptively upregulated upon
NOP56 depletion (Fig. 3G, H). Strikingly, rapamycin
induced PARP cleavage (Cl PARP) in NOP56 KD H358
but not in control cells (Fig. 4A), indicating that con-
comitant targeting of NOP56 and mTOR caused syn-
thetic lethality. Similar results were observed in NOP56
KD H358 cells that were treated with the AKT inhibi-
tor AZD5363 (Fig. S4A, B). Knockdown of Raptor and
Rictor, key components of the mTORC1 and mTORC2,
respectively, significantly suppressed the viability of
NOP56 KD H358 cells despite to differential extent (Fig.
S4C, D). Moreover, whereas individual S6 and eIF4E only
partly contributed to the viability of NOP56 KD H358
cells, concomitant inhibition of S6 (siRNA) and eIF4E
(siRNA and Briciclib, an eIF4E inhibitor) led to signifi-
cantly enhanced anti-proliferative effect (Fig. 4B-G; Fig.
S4E) and largely recapitulated the impact seen by mTOR
inhibition with rapamycin (Fig. 4A, Fig. S4F), gauged
by the extent to which apoptotic markers (CI-PARP)
were induced in NOP56 KD H358 cells (Fig. 4F). These
results interrogate an important role for mTOR to relay
the IRE1α-mediated UPR signaling in NOP56-depleted
KRAS-mutant lung cancer.

The UPR is a double-edged sword, as its outcome flips
from adaption to apoptosis when malfunctional UPR, a
condition at which stress stimuli are overwhelming or the
UPR signal cannot be properly relayed [38, 39]. We thus
assumed that the observed synthetic lethality of NOP56
and mTOR inhibition might be enabled due to malfunc-
tional UPR. Indeed, co-targeting NOP56 and mTOR
resulted in synergistic effects that not only increased the
expression of IRE1α and p-IRE1α, indicative of hyperac-
tive UPR signals, but also upregulated p-JNK, FOXO3A
and BIM, a BH3 only protein and key mediator of apop-
totic balance (Fig. 4H). Consistent with their pro-apop-
totic roles, this increase of the JNK-FOXO3a-BIM axis
was accompanied by PARP cleavage (Cl-PARP) and

significantly greater apoptotic cell death in NOP56 KD
H358 and H460 cells compared to control cells (Fig. 4H,
I). Importantly, IRE1α KD (siRNA) precluded the cyto-
toxicity of combined NOP56 and mTOR inhibition, evi-
denced by decreased levels of p-JNK, FOXO3a, BIM,
Cl-PARP and of apoptotic populations in NOP56 KD
H358 and H460 treated with rapamycin (Fig. 4H, I). Simi-
larly, inhibiting JNK activity by the inhibitor SP600125
dampened the efficacy of rapamycin in NOP56 KD H358
and H460 cells (Fig. 4J). Thus, IRE1α-mediated UPR acti-
vates mTOR, which provides a survival signal for NOP56
KD KRAS-mutant cancer cells; conversely, mTOR inhi-
bition leads to overwhelmed UPR and promotes apop-
totic cell death by activating the JNK-FOXO3A-BIM axis
(Fig. 4K).

NOP56 and mTOR converge on a metabolic liability
in KRAS‑mutant lung cancer
Next, we asked if the synthetic lethality of co-targeting
NOP56 and mTOR is a result of unresolvable metabolic
stress. Indeed, rapamycin sensitivity of NOP56 KD H358
and H460 cells was highly correlated with ROS levels
(Fig. 5A, B), and ROS scavenge by NAC significantly
compromised the cytotoxicity of rapamycin (Fig. 5B),
highlighting a causative link between ROS and rapamy-
cin-induced apoptosis in NOP56 KD cells.

These results uncover a novel homeostatic mechanism
of metabolic stress mediated by NOP56 and validate an
unexpected synthetic lethality by targeting NOP56 and
mTOR that aggregate a metabolic liability in KRAS-
mutant lung cancer (Fig. 5C).

NOP56 downregulation plus rapamycin potently
suppresses in vivo tumor growth of KRAS‑mutant lung
cancer
Finally, we investigated in vivo efficacy of co-targeting
NOP56 and mTOR. In a xenograft model from KRAS-
mutant H460 cells, NOP56 knockdown (shNOP56) only
mildly inhibited tumor growth compared to control

Fig. 4 Synthetic lethality by targeting NOP56 and mTOR in KRAS‑mutant lung cancer cells. A, Immunoblots of H358 cells expressing scramble
control or NOP56‑specific shRNAs after treated with rapamycin (1 μM) for 24 h. B‑G H358 cells expressing scramble control or shNOP56‑specific
shRNAs were transfected with control siRNAs or the indicated siRNAs specifically targeting S6, eIF4E, alone and in combination. The cells were
then subjected to immunoblots (B, D, F) and viability assay (C, E, G) 72 h post‑transfection. Data are presented as mean ± SD (n = 3). H, H358 cells
expressing scrambled control or NOP56‑specific shRNAs were transfected with IRE1α-specific or control siRNAs for 48 h, followed by treatment with
rapamycin (1 μM) for 24 h before immunoblotting. I, H358 cells expressing control or NOP56-specific shRNAs were transfected with IRE1α-specific
or control siRNAs for 24 h, followed by treatment with rapamycin (5 μM) for 72 h before apoptosis assay. Data are presented as mean ± SD
(n = 3). *p < 0.05, ***P < 0.001 and ****P < 0.0001 by two‑way ANOVA with Tukey’s multiple comparisons test. J, H358 cells expressing control or NOP56-specific shRNAs were preincubated overnight with vehicle (DMSO) or the JNK inhibitor SP600125, followed by treatment with rapamycin for 72 h before apoptosis assay. Data are presented as mean ± SD (n = 3). **p < 0.01, ***P < 0.001 and ns P>0.05 by two‑way ANOVA with Tukey’s
multiple comparisons test. K, Proposed model of cellular gauge for IRE1α‑regulated UPR. In KRAS‑mutant cancer cells, intact NOP56 keeps ROS in
check so that IRE1α‑regulated UPR is minimal (basal level; left). Intermediate levels of IRE1α‑regulated UPR ensue from NOP56 depletion, which
activates p38‑AKT/mTOR and promotes cell survival (middle). At “dangerous” level of ROS, IRE1α‑regulated UPR initiates JNK‑dependent apoptosis
(right)

(See figure on next page.)

Page 12 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

Fig. 4 (See legend on previous page.)

Page 13 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

shRNA (shScrambled), as did rapamycin (Fig. 6A).
However, the outcome of concomitant targeting of
NOP56 and mTOR (shNOP56 plus rapamycin) was
superior to that achieved by shNOP56 or rapamycin
alone, leading to far more effective and potent suppres-
sion of xenograft tumor growth (Fig. 6A, B). Residual
tumors (after 3-week treatment) from the combina-
tion group (shNOP56 plus rapamycin) were typically
tiny and significantly differed from those of the other
treatment groups (Fig. 6C). Immunohistochemical

(IHC) analysis revealed that the anti-tumor efficacy
of combined treatment with shNOP56 and rapamycin
was paralleled by marked decrease of mTOR activity
(p-AKT, p-mTPOR, p-S6) and increase in apoptosis
(Caspase-3) in the residual tumors (Fig. S5A).

Similar results were obtained from H358 xenografts
(Fig. 6D, E) and a patient-derived xenograft (PDX) model
established from the primary KRAS-mutant PF139 cells
(Fig. 6F-I). In both models, NOP56 KD sensitized H358
and PF139 xenograft tumors to rapamycin, leading to

Fig. 5 NOP56 and mTOR converge on a metabolic liability in KRAS‑mutant tumor growth. A, Apoptosis assay of H358 and H460 cells expressing
control or NOP56‑specific shRNAs after treatment with rapamycin (5 μM) for 72 h. Data are presented as mean of three independent experiments
(n = 3). *p < 0.05, ****P < 0.0001 and ns P>0.05 by two‑way ANOVA with Tukey’s multiple comparisons test. B, H358 and H460 cells expressing control
or NOP56‑specific shRNAs were treated with rapamycin (5 μM) for 24 h. Cells were then washed, incubated with H2DCFDA for 30 min, and analyzed
by flow cytometry. Quantification of relative ROS levels was shown in the right. C, A working model for the function of NOP56 in KRAS‑mutant
cancers

Fig. 6 NOP56 knockdown plus rapamycin inhibits KRAS‑mutant tumor growth. A, Growth curve of xenograft tumors derived from H460 cells
expressing either a control or an shRNA against NOP56 (shNOP56a). Rapamycin (0.1 mg/kg) was administrated i.p. for 3 weeks (5 days/week). Data
are shown as mean ± SD). ***P < 0.001, *P < 0.05 and ns (P>0.05) by two‑way ANOVA with Tukey’s multiple comparisons test. B, Relative tumor
volume of H460 xenograft tumors after the treatment for 3 weeks. C, Weights of H460 xenograft tumors after the treatment for 3 weeks. ***P < 0.001 by one‑way ANOVA with Tukey’s multiple comparisons test. D, Growth curve of xenograft tumors derived from H358 cells expressing either a control or an shRNA against NOP56 (shNOP56a). **P < 0.01 by two‑way ANOVA with Tukey’s multiple comparisons test. E, Kaplan‑Meier survival curve of mice with H358 xenografts from the experiment in D. F, Growth curve of PDX tumors derived from primary KRAS‑mutant PF139 lung cancer cells expressing either control or NOP56‑specific shRNAs. Data are shown as mean ± SD). ***P < 0.001 and *P < 0.05 by two‑way ANOVA with Tukey’s multiple comparisons test. Immunoblots of PF139 cells expressing NOP56‑specific or scrambled shRNAs was also shown. G, Relative tumor volume of PF139 xenografts after 3 weeks of treatment. H, Weights of PF139 xenograft tumors after treated for 3 weeks. ***P < 0.001, *P < 0.05 and ns P>0.05 by two‑way ANOVA with Tukey’s multiple comparisons test. I, H&E and IHC of p‑AKT(T308), p‑mTOR(S2448), p‑S6(S235/236), Ki67 and
Caspase‑3) in PF139 xenograft tumors after the treatment. Scale bars 100 μm

(See figure on next page.)

Page 14 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

Fig. 6 (See legend on previous page.)

Page 15 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

potent suppression of tumor growth (Fig. 6D, F-H) and
significant improvement of mouse survival (Fig. 6E). IHC
of the PF139 residual tumors revealed that NOP56 deple-
tion plus rapamycin strikingly suppressed tumor cell pro-
liferation (Ki-67) and dampened mTOR activity (p-AKT,
p-mTOR and p-S6), but increased Caspase-3 cleavage
(Fig. 6I), which is consistent with the in vitro results
(Figs. 4, 5) and our observations on H460 xenografts (Fig.
S5A). Notably, rapamycin showed little beneficial effects
in NOP56 KD KRAS wild-type H1703 xenografts (Fig.
S5B, C), which mirrors the in vitro data and reinforces
the selective activity of co-targeting NOP56/mTOR in
KRAS-mutant lung cancer.

Together, these results support a model that NOP56
downregulation induces a metabolic vulnerability to
mTOR inhibition, which presents a new and rational
strategy for treating KRAS-mutant lung cancer.

Discussion
In the present study, we have uncovered a new and
unanticipated mechanism by which NOP56 and mTOR
signaling cooperate in metabolic stress response in
KRAS-mutant lung cancer. We show that NOP56 sup-
presses ROS, and its depletion induces synthetic lethal
susceptibility to inhibition of mTOR that is otherwise
essential for counterbalance of the resurge of cytotoxic
ROS evoked by NOP56 downregulation. We also dis-
cover that mTOR activation is driven by IRE1α-mediated
UPR via p38 MAPK. These findings support a model that
NOP56 plays a role in the surveillance of ROS homeosta-
sis and suggest that concomitant blockage of NOP56 and
mTOR signaling has the potential to selectively target
KRAS-mutant lung cancer. As multiple mTOR inhibitors
(e.g., rapamycin and everolimus) are clinically approved
drugs, our observations have immediate translational
significance.

Despite decades-long steady efforts, therapeutic tar-
geting of KRAS-mutant cancers has remained an over-
arching challenge in clinical oncology [3]. A promising
strategy to target KRAS-driven tumors is to exploit can-
cer cell vulnerabilities contextually co-opted by mutant
KRAS, in light of the concept that mutant KRAS alter
physiological biochemical networks and induces cellu-
lar stresses, rendering KRAS-mutant cancer particularly
susceptible to inhibition of stress-remedy mechanisms
[12–14]. Empowered by CRISPR- and shRNA-based
functional genomics, a plethora of novel factors required
for KRAS-mutant cancer cells have been identified [12–
16, 26], although the long-sought-after universal syn-
thetic lethal targets for KRAS-driven pan-cancers are still
at large. By implementing integrated analysis of func-
tional genomic datasets (n = 5) derived from shRNA-
and CRISPR-based screens [12, 16, 26], we revealed

that NOP56 confers a metabolic requirement for
KRAS-mutant cancer by regulating ROS homeostasis.
A functional link between NOP56 and mutant KRAS is
supported by a multitude of lines of evidence, i.e., the ele-
vated expression of NOP56 in KRAS-mutant tumors, the
prognostic significance of NOP56 expression in patients
with KRAS-mutant but not wild-type cancers, and selec-
tive damage on KRAS-mutant cancer cells incurred by
NOP56 downregulation.

Moreover, our results uncover a reciprocal interaction
of NOP56 and mTOR signaling and suggest that com-
bined inhibition of NOP56/mTOR is a rational strategy
to combat KRAS-mutant cancer. Supporting our find-
ings, mRNA levels of NOP56 significantly correlate with
that of mTOR pathway genes in KRAS-mutant cancer
cells and lung adenocarcinomas, and NOP56 expression
is a predictive marker of sensitivity to mTOR inhibi-
tors in KRAS-mutant but not KRAS-wild-type cancer
cells. Importantly, NOP56 knockdown sensitizes KRAS-
mutant cancer cells to mTOR inhibitors in vitro and
in vivo, which is not true for KRAS-wild-type tumor cells.
Despite potential toxicological challenges as ribosome
biogenesis is also an important physiological process,
modulation of NOP56 activity may afford a therapeu-
tic window for targeted inhibition of mTOR in KRAS-
mutant cancers.

An increasingly growing body of evidence suggests that
oncogenic KRAS signaling rewires metabolic pathways to
meet the energetic and biosynthetic demands of cancer
cells [27–30, 40]. In particular, increased ROS produc-
tion, which has been shown to be functionally required
for KRAS-mediated tumorigenicity [27, 28], is a key
metabolic manifestation associated with KRAS-mutant
cancer cells [27–30]. Since excess ROS is harmful, cancer
cells must leverage ROS levels to favor tumor progression
but prevent cell death [14, 27–30, 41]. Here, we reported,
for the first time, a role for NOP56 in metabolic ROS
response in KRAS-mutant lung cancer. NOP56 is a key
component of box C/D snoRNPs that regulates ribosome
assembly. This process has been shown to be deregulated
in tumors with increased requirement for protein syn-
thesis, providing cancer vulnerabilities for therapeutic
avenues [21–24, 42–45]. Our results are also in line with
previous studies reporting cancer subtype-specific altera-
tions in ribosome assembly and biogenesis processes [44,
45]. In addition, recent evidence suggests that snoRNPs
may also be involved in other processes independent of
their functions in ribosome biogenesis [46]. Future stud-
ies will be necessary to clarify whether the newly identi-
fied metabolic role of NOP56 in KRAS-mutant cancer is
related to its canonical role.

Our finding that IRE1α-mediated UPR connects
the NOP56 function in ROS scavenge with mTOR

Page 16 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

signaling, a master regulator of cellular metabolism
[47], provides mechanistic insights about the syn-
thetic lethality of co-targeting NOP56 and mTOR,
which is supported the observation that challenges
to ribosome biogenesis result in acute loss of pro-
teostasis [48]. IRE1α-mediated UPR, whilst initially
protective, turns to be pro-apoptotic if ROS-induced
metabolic stress is prolonged and persists [38, 39].
We further reveal that the overwhelming metabolic
stress incurred by NOP56 depletion and mTOR inhi-
bition activates the JNK-BIM axis, a stress-responsive
pathway that promotes cell-cycle arrest and apoptosis
[37]. The observation that UPR is a key component
of the homeostatic mechanism in response to ROS
resurge evoked by NOP56 knockdown is consist-
ent with the notion that UPR plays an import role in
homeostasis regulation including ROS and that chal-
lenges to ribosome biogenesis result in acute loss
of proteostasis [49]. As metabolic ROS is causally
linked to mutant KRAS-induced tumorigenicity and
requires homeostatic mechanisms to maintain ROS
levels within a threshold favorable for tumor devel-
opment [14, 27–30, 41], the identification of NOP56
and mTOR converging on a role as ROS scavengers
reveals an unanticipated metabolic vulnerability in
KRAS-mutant cancers.

Conclusion
In summary, we have uncovered an unexpected role
for NOP56 in the surveillance of metabolic ROS in
KRAS-mutant lung cancer. We have also revealed a
novel synthetic lethality between NOP56 depletion
and mTOR inhibitors that occurs by impeding the
homeostatic mechanism of ROS in KRAS-mutant can-
cer cells. Moreover, we have demonstrated that mTOR
activation upon NOP56 depletion is driven by IRE1α-
mediated UPR. These results shed light on the mecha-
nisms underlying KRAS-induced metabolic rewiring,
reveals an unanticipated metabolic vulnerability in
KRAS-mutant lung cancer, and suggest a new ration-
ale for the treatment of the disease. Because KRAS
alterations are implicated in a broad spectrum of
human malignancies, our findings may also be appli-
cable to other lineages of cancer with high frequencies
of KRAS alterations.

Abbreviations
GSEA: Gene Set Enrichment Analysis; IRE1α: Inositol‑requiring enzyme 1α;
KRAS: Kirsten rat sarcoma viral oncogene homolog; mTOR: Mechanistic target
of rapamycin; NOP56: Nucleolar protein 5A; PARP: Poly (ADP‑ribose) polymer‑
ase; RNAi: RNA interference; ROS: Reactive oxygen species; TCGA : The Cancer
Genome Atlas; UPR: Unfolded protein response.

Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s13046‑ 022‑ 02240‑5.

Additional file 1: Figure S1. NOP56 knockdown inhibits proliferation of
KRAS‑mutant cancer cells. A, Immunoblots of KRAS‑mutant and KRAS‑wild
type cancer cells that were transfected with NOP56‑specific siRNAs (si‑
NOP56) or scramble control siRNAs (si‑Control). B, KRAS mutant and KRAS
wild type cancer cells were transfected with control siRNAs or NOP56‑
specific siRNAs. Cell viability was determined 72 h post transfection. Data
are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***P < 0.001, ****P < 0.0001 and ns P>0.05 by two‑way ANOVA with Tukey’s multiple
comparisons test. C, NOP56 is not a biomarker of survival in patients with
KRAS‑wild‑type lung adenocarcinoma (LC), pancreatic cancer (PC) and
colon cancer (CC). Kaplan–Meier survival analyses of patient cohorts
in TCGA were stratified by the optimal cut‑off value of the mRNA level
of NOP56. D, Gene set enrichment analysis (GSEA) of a TCGA cohort of
patients with KRAS‑mutant lung (n = 141), pancreatic (n = 133) and colon
cancer (n = 170). Figure S2. Stable expression of NOP56-specific shRNAs
activates IRE1α‑mediated UPR. A, Immunoblots of H358 and H460 cells
expressing scrambled control or NOP56 shRNAs. B, Immunofluorescence
of H358 and H460 cells that express scrambled control or NOP56 shRNAs.
The NOP56 signal is indicated by arrowheads. C, The cell viability curve of
H358 and H460 cells expressing scramble control shRNA or the NOP56‑
targeted shRNAs was measured at the indicated time points. D, Clongenic
assay of H358 and H460 cells expressing scramble control or NOP56‑tar‑
geted shRNAs. Quantification of clongenic assay were shown underneath.
Data are presented as mean ± SD (n = 3). E, Growth inhibition of H358
and H460 cells expressing control shRNA or NOP56‑targeted shRNA (3000
cells/well) treated for 72 h with the indicated doses of an IRE1α inhibitor
(4μ8C). Data are presented as mean ± SD (n = 3). F, Apoptosis assay of
H460 cells expressing scrambled control or NOP56‑targeted shRNAs
after transfection with IRE1α-specific or control siRNAs for 72 h. Data are
presented as mean ± SD (n = 3). ***P < 0.001 and ns P>0.05 by two‑way
ANOVA with Tukey’s multiple comparisons test. Figure S3. NOP56 KD
renders KRAS‑mutant lung cancer cells susceptible to mTOR inhibition. A,
Bar graphs illustrating the change of sensitivity to different inhibitors in
H460 cells after NOP56 knockdown. Data are presented as IC50 values of
the indicated inhibitors in H460 cells expressing scramble control shRNAs
compared to IC50 in H460 cells expressing NOP56‑targeted shRNAs. Data
are shown as mean (n = 2). B, Viability assay of H460 and H358 cells
expressing control shRNA or NOP56‑targeted shRNA (3000 cells/well)
after treated for 72 h with the indicated doses of PI3K inhibitor (LY294002)
and AKT inhibitor (AZD5363). Data are presented as mean ± SD (n = 3).
C, Viability assay of H460 and H358 cells expressing control shRNA or
NOP56‑targeted shRNA (3000 cells/well) after treated for 72 h with the
indicated doses of BiP inhibitor (HA15) and ER stress inducer (bortezomib).
Data are presented as mean ± SD (n = 3). D, Immunoblots of KRAS‑mutant
(H358, H460) and wild‑type (H1703, H520) cells expressing NOP56‑specific
sgRNAs. E, Viability assay of the cells expressing control or NOP56‑
specific sgRNAs after treated with rapamycin for 72 h. Data are shown
as mean ± SD (n = 3). F, NOP56 is negatively correlated with PI3K/AKT/
mTOR pathway genes (PI3KCA, PDPK1, PIK3R1) in KRAS‑mutant lung cancer
patients. Pearson and Spearman coefficient and significance (p‑value)
are analyzed using R software (Cor.test function). G, Immunoblots of
H460 and H1703 cells expressing control or NOP56-targeted shRNAs. H, I,
Viability assay of KRAS‑mutant (H) and wildtype (I) cancer cells expressing
control or NOP56‑specific siRNAs after treated with rapamycin. The assay
was performed 72 h after drug treatment (96 h after siRNA transfection).
Figure S4. NOP56 KD activates and induces dependence on the mTOR
pathway in KRAS‑mutant cancer cells. A, Immunoblots of H358 cells
expressing control or NOP56‑targeted shRNAs after treated with the AKT
inhibitor (AZD5363) for 24 h. B, Clongenic assay of H358 cells express‑
ing control or NOP56‑specific shRNAs after treated with indicated doses
of AZD5363. Representative images are shown. C, D, Immunoblots (C)
and viability assay (D) of H358 cells expressing control or NOP56-specific
shRNAs after transfected with raptor‑ or rictor‑specific or control siRNAs for
72 h. Data are presented as mean ± SD (n = 3). E, Clongenic assay of H358
and H460 cells expressing control shRNA or NOP56‑specific shRNAs after

https://doi.org/10.1186/s13046-022-02240-5

Page 17 of 18Yang et al. J Exp Clin Cancer Res (2022) 41:25

treatment with indicated doses of eIF4E inhibitor (Briciclib). Representative
images are shown. F, Immunoblots of H460 cells expressing control or
NOP56‑target shRNAs after treated with rapamycin (1 μM) for 24 h. Figure
S5. In vivo activity and selectivity of co‑targeting NOP56 and mTOR
in KRAS‑mutant lung cancer. A, H&E and IHC analysis of p‑AKT(T308),
p‑mTOR(S2448),p‑S6(S235/236), Ki67 and Caspase‑3) in residual H460
xenograft tumors after the indicated treatment. Scale bars 100 μm. B,
Tumor volume of H1703 xenografts in immunocompromised (NSG) mice.
H1703 cells were transduced with either a control or an shRNA against
NOP56 (shNOP56a). Tumors were measured every 5 days with a caliper. C,
Kaplan‑Meier survival curve of mice harboring H1703 xenografts from the
experiment shown in B.

Additional file 2: Table S1. Cell lines used in this study. Table S2. Inhibi‑
tors used for synthetic lethal chemical screens. Table S3. Antibodies used
in this study. Table S4.KRAS synthetic lethal (SL) genes.

Acknowledgements
We gratefully acknowledge Christelle Dubey (Division of Thoracic Surgery,
Inselspital, Bern University Hospital) for technical support, especially with ani‑
mal studies and CRISPR‑based knockout of NOP56. We thank the West‑Ger‑
man Biobank Essen (WBE) for the collaboration in establishment of the PF139
and PF526 lung cancer cells. The Translational Research Unit at the Institute of
Pathology, University of Bern is acknowledged for assistance of IHC staining.

Authors’ contributions
ZY, SQL designed and performed the experiments, analyzed the data and
wrote the manuscript. LZ, HY performed the experiments and analyzed the
data. TMM, BH, YG, BZ, CC and WW analyzed the data and edited the manu‑
script. PD and GJK provided conceptual inputs, analyzed the data and edited
the manuscript. RAS provided financial support and edit the manuscript. RWP
conceived the project, supervised the study and wrote the paper. All authors
read and approved the final version of the manuscript.

Funding
This study was supported by a grant from Swiss National Science Founda‑
tion (SNSF #310030_192648; to R‑W. Peng) and PhD fellowships from China
Scholarship Council (ZY, LZ, YG).

Availability of data and materials
All data generated or analysed during this study are included in this published
article and its supplementary information files.

Declarations

Ethics approval and consent to participate
The establishment of PF139 and PF526 cells was approved by the Ethics Com‑
mittee of the University Hospital Essen (#18–8208‑BO), Germany, with written
consents obtained from the patients. The study was performed in accord‑
ance with the Declaration of Helsinki. Mouse studies were approved by the
Veterinary Office of Canton Bern, Switzerland, and conducted in accordance
with Institutional Animal Care.

Consent for publication
Not applicable.

Competing interests
The authors declare no competing interests.

Author details
1 Division of General Thoracic Surgery and Department of BioMedical Research
(DBMR), Inselspital, Bern University Hospital, University of Bern, Murtenstrasse
28, 3008 Bern, Switzerland. 2 Current address: University of Massachusetts
Medical School, Worcester, MA 01605, USA. 3 Current address: Department
of Thoracic Surgery, Shanghai Chest Hospital, Shanghai Jiao Tong Univer‑
sity, Shanghai 200030, China. 4 Department of Thoracic Surgery, University
Medicine Essen – Ruhrlandklinik, University Duisburg‑Essen, Essen, Germany.
5 Department of Thoracic surgery, Fujian Medical University Union Hospital,
Fuzhou City, Fujian, China. 6 Thoracic Surgery Department 2, Hunan Cancer

Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine,
Central South University, Changsha, Hunan, China.

Received: 23 May 2021 Accepted: 1 January 2022

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Metabolic synthetic lethality by targeting NOP56 and mTOR in KRAS-mutant lung cancer

Abstract
Background:
Method:
Results:
Conclusions:
Background
Materials and methods
Cell culture and reagents
Cell viability and clonogenic survival assay
Apoptosis assays
Gene silencing by small interfering (siRNA), short hairpin RNAs (shRNA) and single-guide RNAs (sgRNA)
Quantitative real-time PCR (qRT-PCR)
Immunoblotting, immunohistochemistry and immunofluorescence
In vivo mouse study
Public databases
Statistical analysis
Results
NOP56 confers a metabolic dependency by regulating ROS homeostasis in KRAS-mutant lung cancer
NOP56 suppression evokes IRE1α-mediated UPR to mitigate oxidative stress
IRE1α-mediated UPR fuels mTOR signaling via p38 MAPK
Synthetic lethality by targeting NOP56 and mTOR in KRAS-mutant lung cancer
NOP56 and mTOR converge on a metabolic liability in KRAS-mutant lung cancer
NOP56 downregulation plus rapamycin potently suppresses in vivo tumor growth of KRAS-mutant lung cancer
Discussion
Conclusion
Acknowledgements
References

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