cancers
Review

Targeting RB1 Loss in Cancers
Paing Linn 1,2 , Susumu Kohno 1 , Jindan Sheng 1 , Nilakshi Kulathunga 1 , Hai Yu 1 , Zhiheng Zhang 1 ,Dominic Voon 3 , Yoshihiro Watanabe 4 and Chiaki Takahashi 1, *
1

2
3

4

*



Citation: Linn, P.; Kohno, S.; Sheng,J.; Kulathunga, N.; Yu, H.; Zhang, Z.;
Voon, D.; Watanabe, Y.; Takahashi, C.
Targeting RB1 Loss in Cancers.
Cancers 2021, 13, 3737.
https://doi.org/10.3390/cancers
13153737
Academic Editors: Paola Indovina,
Francesca Pentimalli andAntonio Giordano
Received: 18 May 2021
Accepted: 21 July 2021
Published: 25 July 2021

Division of Oncology and Molecular Biology, Cancer Research Institute, Kanazawa University,
Kanazawa 920-1192, Japan; dr.painglinn@gmail.com (P.L.); skohno@staff.kanazawa-u.ac.jp (S.K.);
jindan000000@gmail.com (J.S.); nilakshi.kulathunga@gmail.com (N.K.); adam9426@hotmail.com (H.Y.);zhangzhiheng930713@yahoo.co.jp (Z.Z.)
Yangon General Hospital, Yangon, Myanmar
Institute of Frontier Sciences Initiative, Kanazawa University, Kanazawa 920-1192, Japan;
dvoon@staff.kanazawa-u.ac.jp
iCREK, Kanazawa University Hospital, Kanazawa 920-8641, Japan; yoshi.watanabe@staff.kanazawa-u.ac.jp
Correspondence: chtakaha@staff.kanazawa-u.ac.jp; Tel.: +81-76-264-6750; Fax: +81-76-234-4521

Simple Summary: Irreversible defects in RB1 tumor suppressor functions often predict poor outcomes in cancer patients. However, the RB1-defecient status can be a benefit as well for them, as it
generates a variety of vulnerabilities induced through the upregulation of RB1 targets, relief fromfunctional restrictions due to RB1 binding, presence of genes whose inactivation cause synthetic
lethality with RB1 loss, or collateral synthetic lethality owing to simultaneous loss of neighboring genes.
Abstract: Retinoblastoma protein 1 (RB1) is encoded by a tumor suppressor gene that was discovered
more than 30 years ago. Almost all mitogenic signals promote cell cycle progression by braking on thefunction of RB1 protein through mono- and subsequent hyper-phosphorylation mediated by cyclinCDK complexes. The loss of RB1 function drives tumorigenesis in limited types of malignancies
including retinoblastoma and small cell lung cancer. In a majority of human cancers, RB1 function
is suppressed during tumor progression through various mechanisms. The latter gives rise to theacquisition of various phenotypes that confer malignant progression. The RB1-targeted molecules
involved in such phenotypic changes are good quarries for cancer therapy. Indeed, a variety ofnovel therapies have been proposed to target RB1 loss. In particular, the inhibition of a number of
mitotic kinases appeared to be synthetic lethal with RB1 deficiency. A recent study focusing on aneighboring gene that is often collaterally deleted together with RB1 revealed a pharmacologicallytargetable vulnerability in RB1-deficient cancers. Here we summarize current understanding on
possible therapeutic approaches targeting functional or genomic aberration of RB1 in cancers.
Keywords: RB1; E2F; chromatin instability; synthetic lethality; collateral lethality

Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
The discovery of RB1 gene had paved an avenue toward understanding the complicated functions of the human genome in preventing cells from carcinogenesis [1,2]. RB1gene was thought to be mechanistically involved in the initiation of retinoblastoma as
its inactivation following genetic mutation or deletion was prevalently identified in thepedigrees of hereditary cases [3]. This notion was later experimentally proven by the
induction of retinoblastoma in mice following the ablation of multiple Rb family members [4]. Together with the presence of other cancer driving mutations in genes such as
Trp53, RB1 loss can provoke carcinogenesis from various types of tissue. This suggests thatRB1 may in fact be involved in the initiation and/or progression of previously unexpectedvarieties of cancer [5]. Of note, RB1 inactivation is detected in close to 90% of sporadic
small cell lung cancers (SCLC), suggesting a contribution to their initiation, while its germ

Cancers 2021, 13, 3737. https://doi.org/10.3390/cancers13153737

https://www.mdpi.com/journal/cancers

Cancers 2021, 13, 3737

2 of 14line mutation also often predisposes to SCLC [6]. Reportedly, RB1 inactivation occurs in
20~40% of osteosarcoma cases, yet it is thought to contribute to tumor progression based on
pathological examination and linkage with unfavorable outcome [7,8]. RB1 reconstitutionin RB1-deficient osteosarcoma-derived cells does not always completely attenuate theirmalignant phenotypes, presumably due to coexisting functional aberration in Trp53 tumor
suppressor or other driver mutations [9,10]. These findings also indicate that RB1 mayexert varied functions depending on cancer tissue type and timing during the course oftumor development.
Similar to osteosarcoma, RB1 is suggested to be inactivated during their progression ina majority of cancer types [7]. One of the best examples is prostate cancer. In primary/nonmetastatic prostate cancers, RB1 deletion is identified with less than 10% prevalence, but
this rises to higher than 30% in those attaining metastatic and/or castration-resistantphenotypes [11]. In addition to prostate cancer, many common types of cancers, including
non-small cell lung cancer (NSCLC), breast cancer, bladder cancer, chronic myelogenous
leukemia (CML), high-grade glioma, esophageal cancer, head and neck cancer and sarcoma,
are thought to gain RB1 deficiency during their malignant progression [7].
The roles of RB1 in suppressing malignant progression of prostate cancer may not
be solely explained by its primary function to constrain cell cycle progression. For example, RB1 controls the transcription of androgen receptor (AR) through the E2F family oftranscription factors, hence its inactivation could enhance the refractoriness to endocrine
therapy [12]. Moreover, the depletion of RB1 from RB1-proficient prostate cancer cells
promoted gain of stem cell-like properties through increased expression of interleukin-6(IL-6) and lysyl oxidase (LOX) [13]. Similarly, RB1 inactivation in breast cancers enhances
stem cell-like behaviors and malignant progression through IL-6 and following activationof signal transducer and activator of transcription 3 (STAT3) activation [14–18]. RB1 loss
in breast cancer cells activates fatty acid oxidation (FAO) and induces following Jun kinase (JNK) activation. Activated JNK then stimulates IL-6 production leading to gain of
undifferentiated characters which are mediated by cell-autonomous STAT3 activation [14].
Later, the enhanced FAO following RB1 loss was explained by AMP-activated protein
kinase (AMPK) activation and subsequent phosphorylation (downregulation) of acetylCoA carboxylase (ACC) [16]. Accordingly, the blockade of IL-6 by neutralizing antibody
suppressed breast cancer progression is induced following RB1 inactivation [14]. Lossof Trp53 in mice generates well differentiated soft tissue sarcoma with low prevalence;
additional loss of Rb1 converts this to undifferentiated pleomorphic type [17]. An increasein lineage plasticity has been observed in mouse Trp53-null osteosarcoma cells following
Rb1 deletion [19]. Identification of molecules mediating RB1 functions in controlling undifferentiated characters of tumor cells may endow us new tools to target cancer stem
cells [18]. Taken together, in addition to targeting aberrant cell cycle progression, functional
suppression of the proteins aberrantly expressed as a result of RB1 inactivation may also
be beneficial in cancer treatment.
This review aims to firstly shed light on such gene products and then focus on genes
whose inactivation might exhibit synthetic lethality when RB1 is simultaneously inactivated.
Finally, this review will discuss putative approaches to treat RB1-deficient cancers bytargeting comparatively large-scale chromosomal aberration involving the RB1 loci.
2. Targeting Gene Products Upregulated Following RB1 Inactivation
The RB1/E2Fs/DPs complex primarily suppresses target gene transcription by recruiting histone deacetylase (HDAC). Loss of RB1 function liberates its most important
counterpart, E2Fs, and these primarily transactivate target genes by recruiting histone
acetylase (HAT). The target genes of the “transactivating” E2Fs (E2F1, E2F2 and E2F3a)contain a number of genes enrolled in cell cycle progression, DNA synthesis and replication,
DNA damage repair and apoptosis [20]. In line with this, the deletion of E2f1 suppressed
pituitary and thyroid tumorigenesis and prolonged the life span in Rb1-heterozygous
mice [21]. Likewise, the loss of E2f2 or E2f3 in MMTV-Myc transgenic mice resulted in theCancers 2021, 13, 3737

3 of 14cessation of mammary carcinogenesis [22]. E2F1 is frequently overexpressed in metastaticmelanoma, and inhibition of E2F1 induced apoptosis and cellular senescence [23]. In
specific types of cancer, higher expression of some of E2F family members correlates withadvanced stage and poor prognosis [24]. Therefore, targeting E2Fs may yield certain
benefits in cancer therapy. The E2F inhibitor HML006474 was designed based on a virtual
screening of compounds that possibly inhibit DNA binding of the E2F4/DP2 complex.
The E2F4-null cells were less sensitive to this compound compared to wild type, which
supports the specificity of its action [25]. However, to date, no small molecules specificallytargeting any of transactivating E2Fs have been described nor tested in clinical trials.
As E2Fs target a wide variety of genes that could promote or suppress tumor development, it is much safer to consider strategies to target individual E2F target. As numerous
review articles have already expounded on this point, this article picks up only a few. In
particular, although cyclin E and cyclin dependent kinase 2 (CDK2) are representative
targets of E2Fs during G1/S, and inhibitors to CDK2 are undergoing clinical trials [26],the efficacy of these agents and their dependency on the presence of intact RB1 remains ascantly addressed topic.
E2Fs target numerous genes involved in replication control, nucleotide synthesis,
DNA damage response/repair, chromatin structure and mitotic progression. RB1 inactivation induces chromosomal instability (CIN), at least partially, by upregulating spindle
assembly checkpoint protein mitotic arrest deficient 2 (MAD2). Overexpressed MAD2 may
contribute to induced aneuploidy and stabilization of erroneous attachment of mitotic
spindles [27,28]. Dysregulated expression of MAD2 is correlated to cancer progression
and poor prognosis [29]. A small molecule, M2I-1, targeting MAD2-CDC20 interaction has
been developed [30]. A report suggested that M2I-1 sensitizes cancer cells to anti-mitotic
reagents [31] but, thus far, no report has tested its efficacy in RB1-deficient context. Manyof mitotic kinases are upregulated following E2F dysregulation and inhibition of some ofthem has been indicated to be synthetically lethal with RB1 inactivation (see below).
Besides MAD2, numerous possibly druggable molecules have been indicated to be
involved in the RB1 loss-induced CIN [32]. Aurora A and polo-like kinase 1 (PLK1) are
upregulated following RB1 depletion, which may lead to aneuploidy or other mitotic
abnormalities, further causing malignant progression [33]. Synthetic inhibitors to thesekinases are now in clinical trials (Table 1). Mis-localization of CAPD3/condensin II has
been proposed to mediate centromere dysfunction induced by RB1 loss [34]. However,synthetic inhibitors to this molecule are under development and it is not clear whether
such inhibitors could exhibit anti-tumor activity.
In addition to mitotic catastrophe, replicative catastrophe induced by abnormallyenhanced cell cycle progression induced by gain of function by E2Fs could provide a certain
vulnerability especially upon chemotherapy or radiation therapy [35]. The observationthat RB1 inactivation in thyroid calcitonin-producing (C) cells or fibroblastic cells induced
DNA damage response followed by senescence supports this speculation [36].
As alluded to the introduction, mostly in an E2F-dependent manner, RB1 status affects
the milieu surrounding tumor cells including chemokine/cytokine secretion, extracellularmatrix, immune cells or remote organs to be metastasized. The molecules mediating suchfunctions of RB1 could serve as good therapeutic targets [37,38]. C-C motif chemokine
2 (CCL2) appeared to be secreted from RB1-deficient breast cancer cells, thereby suchtumor cells recruit immunosuppressive cells including myeloid-derived suppressive cells(MDSC), regulatory T cells (Treg) or macrophages, allowing malignant progression following RB1 inactivation. Moreover, blockade of CCL2 signaling in host mice by geneticallydeleting CCR2, encoding the receptor for CCL2, almost completely abrogated mammary
carcinogenesis from MMTV-cre; Rb1flox/flox mice [14,38]. RB1 inactivation has been linked
to upregulation of PD-L1 expression through direct interaction with NF-κB protein p65.
Surprisingly, a particular phosphorylated form of RB1 inactivates p65 and therefore attenuates expression of PD-L1 and cIAPs. The peptide mimicking this phosphorylated form of
RB1 improved the therapeutic efficacy in radiotherapy by suppressing radiation-induced

Cancers 2021, 13, 3737

4 of 14

PD-L1 expression [39]. These findings make clear contrast with the enhanced immunogenicity induced by locking RB1 in unphosphorylated form by the treatment with CDK4/6
inhibitor [40], and suggest that RB1 status might affect the efficacy of therapy by immune
checkpoint inhibitors (ICIs).
Table 1. Genes whose inactivation exerts synthetic lethality in the presence of RB1 deficiency,
and representative inhibitors to the gene products and phase of clinical trials. ND: no data. The
information was obtained by searching online accessible catalogues provided by Selleck, Santa Cruz
and Abcam.
Gene

Inhibitor

Phase of Clinical Trial

Aurora A, B

Alisertib
Tozasertib
Barasertib
MNL8054
Danusertib
AT9283
KW-2449
SNS-341
ENMD-2076
BI-847325

III
II
I
I
II
II
I
I
II
I

PLK1

Volasertib
Rigosertib
BI2536

III
III
II

CHK1

AZD7762
MK-8779
PF-477736

I
II
I

CDC25A, B

K-252a
NSC663284
NSC95397

ND
ND
ND

WEE1

Adabosertib

II

TSC1, 2

ND

ND

SKP2

SKPin C1ND

TAF1

CeMMEC-1, 13
BAY299

ND
ND

NUP88

ND

ND

NUP214

ND

ND

PARP

Olaparib
Veliparib
Rucapatib
Iniparib

FDA-approved
III
III
III

SYK

Fostamatinib
R406

III
I

A while ago, an unexpected twist arose from C. elegans studies that indicated lin-35, an
RB1 orthologue, might be placed upstream of Ras signaling during vulval development [41,42].
In cultured cells, including embryonic fibroblasts and thyroid C cells, K-Ras and N-Ras areactivated 5~10 times greater following RB1 loss, and this may promote or suppress tumor
development in a highly context-dependent manner [36,43–47]. It has been estimatedthat mutation of K-Ras or N-Ras in the context of RB1 loss exerts a mitogenic effect thatis 50~60 times greater than that of corresponding wild type Ras in the presence of RB1.
Mechanistically, the RB1-Ras nexus has been explained by farnesylation and geranylgeranylation to anchor cytosolic Ras proteins to lipid bilayers, which are innervated by E2Fs
and SREBPs [36,48], thus this axis is pharmacologically targetable. These findings led

Cancers 2021, 13, 3737

5 of 14researchers to address RB1 functions in cholesterol and then lipid metabolism. ELOVL
fatty acid elongase 6 (ELOVL6) and fatty acid desaturase 1 (SCD1) have been linked to RB1
via E2Fs and SREBPs, which explained the profound impact of RB1 loss on the lipidomic
landscape [49]. The oleic acid, one of the immediate products of ELOVL6 and SCD1, appeared to promote mammary carcinogenesis by stabilizing c-Myc through GPR40 which isa receptor for oleic acid, providing a good rationale to develop antagonists to this receptor
to prevent breast cancer associated with high fat diet in puberty [50].
Although the dependence on E2Fs is still unclear, human retinoblastoma cells highly
express a proto-oncogene spleen tyrosine kinase (SYK) and depend on its expression
for survival. Treatment of genetic retinoblastoma model with synthetic SYK inhibitor
resulted in dramatic suppression of disease progression and prolongation of survival [51].
Furthermore, in retinoblastoma cells, ubiquitin-like, containing PFD and RING finger
domains 1 (UHRF1) and helicase, lymphoid specific (HELLS) are overexpressed, which is
linked to epigenetic activation of SYK [52,53].
3. Partners Other Than E2Fs Liberated upon RB1 Loss
More than 300 proteins have been indicated to directly bind to RB1 [48]. Variedmono-phosphorylation status of RB1 (possibly 14 variations) may increase the complexityof the relationship with specific binding partners [54]. As RB1 is typically not abundantly
expressed in tissues, RB1 may carefully select partners depending on the cellular context.
A number of chromatin modifiers, cyclins and signaling molecules, such as apoptosis
signal-regulating kinase 1 (ASK1) equipped with LxCxE motif, bind to RB1 at the site
also targeted by a number of oncogenic virus products carrying LxCxE motif including
adenovirus E1A, simian virus 40 large T antigen and human papilloma virus E7 [55]. Suchchromatin modifiers include attractive targets of cancer therapy. Notably, 5-azacytidine
targets DNMT1 and has been used for the treatment of myelodysplastic syndrome [56].
Similarly, the utility of pan-HDAC inhibitors has long been evaluated in a variety ofcancers [57]. The Suv39H1 inhibitor chaetocin exhibited significant therapeutic efficacy
against acute myeloid leukemia when combined with other epigenetic drugs that include
suberoylanilide hydroxamic acid (HDAC inhibitor) and JQ-1 (bromodomain inhibitor) [58].
However, how the loss of RB1 impacts the efficacies and sensitivities of these molecules is
not well studied. One notable exception is TAI-1, which targets Hec-1 and exhibits synergy
with multiple chemotherapeutic agents in the treatment of leukemia, breast and liver cancer.
In these settings, the status of RB1 and Trp53 influenced the sensitivity to TAI-1 [59].
SKP2 directly binds to RB1 [60], whereupon RB1 loss liberates SKP2 to promote theproteasomal degradation of p27KIP . SKP2 was initially identified as a cyclin A binding
protein and later found to serve as a component of the SCFSKP2 E3 ubiquitin ligase complex
targeting various substrates containing many of cell cycle regulators, including p27KIP .
Additional loss of Skp2 loci appeared to increase the degree of apoptosis in Rb1-nullizygous
mouse embryonic fibroblasts (MEFs) in an E2F-dependent manner. Similarly, Skp2 loss
increased apoptotic cells in Rb1-deficient pituitary tumor developed in mice, also in an
E2F-dependent manner [61]. Loss of SKP2 stabilizes p27KIP and simultaneously exposes
cyclin A to p27KIP . Additionally, the loss of SKP2 lessens cyclin A activity to attenuate
E2F1 function. Consequently, overactivated E2F1 behaves more proapoptotic rather thanproliferative in the absence of RB1 [61].
Deficiency in terminal differentiation following RB1 inactivation would be anotherpossible target in cancer treatment. Many of the tissue-specific transcription factors, including MYOD, CCAAT enhancer binding protein (C/EBP), glucocorticoid receptor (GR),GATA-binding factor 1 (GATA-1), PU-1, core-binding factor alpha 1 (CBFA-1), pancreas
duodenum homeobox 1 (PDX1), runt-related transcription factor 2 (RUNX2) and nuclear
factor of IL-6 expression (NF-IL6), directly or indirectly collaborate with RB1 to determine
lineage specificity and to induce terminal differentiation [62]. As such, the loss of RB1 promotes undifferentiated behaviors of cancer cells, though the extent to which tissue-specifictranscription factors also contribute to this phenotype is still unclear. Added to this, theCancers 2021, 13, 3737

6 of 14functional recovery of tissue-specific transcription factors by pharmacological approaches
may be difficult.
Yet how the loss of RB1 promotes undifferentiated behaviors of cancer cells might be
at least partially explained by the pivotal roles of RB1 in controlling pluripotency factors
including octamer-binding transcription factor 4 (OCT4) and SRY-box 2 (SOX2), by directly
binding to the regulatory elements for these genes [63]. This study has been done in thecontext of inducible pluripotent stem cells (iPSs). Additionally, as abovementioned, IL-6
and LOX have been proposed to explain the effect of RB1 loss on the malignant progressionof prostate and breast cancer [13,14]. Interference to these machineries may enable cancer
therapy by inducing spontaneous differentiation.
4. Synthetic Lethality with RB1 Inactivation
It is commonly held that loss-of-function mutations or deletions of tumor suppressor
genes are hard to pharmacologically target in cancer therapy. One possible approach totarget cancers with an inactivated tumor suppressor is to find synthetic lethal targets insuch tumor cells [64]. The discovery of synthetic lethal interaction between breast cancer
susceptibility gene 1/2 (BRCA1/2) tumor suppressors and poly ADP-ribose polymerase(PARP) DNA repair protein provided an opportunity to use PARP inhibitors in clinical cancer therapy [65]. With the aim of identifying genes that are in synthetic lethal relationshipwith RB1, RNAi, shRNA, CRISPR/Cas9i or chemical libraries have been often employedon high-throughput screening bases. RB1 mutation contributes to the development of
SCLC (see above). However, it has been difficult to develop methods to directly target RB1
mutation in SCLC due to the lack of ‘druggability’—intrinsic factors favoring the design ofsmall chemical inhibitors. One notable success comes in a pharmacogenomic screening of
RB1-mutant cancer cell lines that identified Aurora A, whose inhibition exhibited cell deathselectively in RB1-mutant SCLC cells [66]. A CRISPR/Cas9-based screening identified
Aurora B whose loss appeared to be synthetic lethal with RB1 loss in SCLC [67]. The latterstudy speculated that inhibition of Aurora B might affect mitotic fidelity in RB1-deficient
cells to a lethal level. Another study using an RNAi library in RB1-knockout lung cancer
cells identified Aurora A and reasoned its mechanism by E2F-mediated upregulation of
microtubule destabilizer Stathmin/OP18. Aurora A inhibition activated Stathmin throughreduction of its phosphorylation, inducing mitotic cell death in RB1-knockout lung cancer
cells [68].
Aurora kinases maintain mitotic fidelity by driving centrosome maturation, spindle
assembly, centrosome bi-orientation and cytokinesis as one member of mitotic kinases.
Many mitotic kinases are aberrantly expressed in RB1-deficient cells in an E2F-dependent
manner and polo-like kinase 1 (PLK1) is one of them [69]. This provides one of the mechanisms whereby RB1 controls mitotic fidelity. Additionally, RB1 inactivation is linked
to chromosomal instability (CIN) at least partially through enhanced replication fork
stalling [69]. Thereby, in specific contexts, RB1 inactivation induces DNA damage response associated with increased activity of ATM, ATR, checkpoint kinase 1 (CHK1) andCHK2 and increased staining for γ-H2AX at the level that subsequently triggers cellularsenescence [36,48,70–72]. A study using a pharmacological screening revealed that thesynthetic CDK4/6 inhibitor strongly antagonizes the toxicity induced by a PLK1 inhibitor
and a CHK1 inhibitor in triple negative breast cancer (TNBC) cells [73]. Conversely, theloss of RB1 highly sensitized TNBC cells to these reagents. Actually, PLK1 and CHK1 wereupregulated in RB1-deficient TNBCs. In the presence of PLK1 inhibitor, RB1-proficient
cells arrest in G2/M, and thus escaped from cell death, but RB1-deficient cells maintainDNA replication and progress to higher aneuploidy and would hence undergo cell deathwhen treated with PLK1 inhibitor. In the presence of CHK1 inhibitor, RB1-deficient cells
exhibited several symptoms of S phase collapse that were absent in RB1-proficient cells [73].
These findings suggest that the unstable mitotic fidelity or dampen DNA damage response
due to the inactivation of RB1, would expose tumor cells to novel vulnerability to agentsCancers 2021, 13, 3737

7 of 14that target these accumulated abnormalities to catastrophically trigger cell death, insteadof resulting in CIN.
SCLC often harbor mutations in RB1 and Trp53, but rare mutation in BRCA1/2 norother genes carrying homologous recombination functions. However, single usage of PARP
inhibitors or combination therapy with another drug have been suggested to be effectivein the treatment of SCLC [74]. Furthermore, in prostate cancers, simultaneous loss of
BRCA2 and RB1 sensitizes tumor cells to PARP inhibitor [75]. In advanced prostate cancer
cases, the co-deletion of RB1 and BRCA2 is frequently found. If even the lack of RB1 alone
increases the sensitivity to PARP inhibitors is not clarified yet.
There are indeed many observations from basic and clinical studies indicating thatloss of RB1 function tend to associate with favorable response to chemotherapy [76]. Moreefficient and prolonged response to chemotherapy was reported in RB1-deficient non-small
cell lung cancers (NSCLC) compared to in their RB1-proficient counterparts. This led theauthors of these studies to propose that RB1 deficiency has resulted in the bypassing ofcertain checkpoints [77]. Similarly, breast cancer cases with disrupted RB pathway tend to
exhibit favorable response to neoadjuvant chemotherapy [78,79]. In TNBC, RB1 deficiency
may better benefit from gamma irradiation and chemotherapy using doxorubicin andmethotrexate rather than RB1-proficient [80]. Similarly, the loss of RB1 function together
with K-Ras mutation predicts favorable response to platinum-based chemotherapy in aparticular type of pancreatic neoplasia [81]. More recently, RB1 loss was identified as one ofpredictive markers of better response to a topoisomerase inhibitor irinotecan [82]. Therefore,
whilst the loss of RB1 function is recognized as a hallmark of an overall poor prognosis
for cancer patients, it may paradoxically provide clinically important vulnerabilities that
could be targeted by genotoxic agents.
The acceleration of cell cycle progression following RB1 inactivation may provide further exploitable vulnerabilities in cancer therapy. Cell division cycle 25 proteins (CDC25s)
are dual phosphatases, of which CDC25A and CDC25B are prominent in the frequency of
their overexpression in cancers. Such a status in TNBC predicts poor prognosis. One of
their most important substrates is cyclin dependent kinase 1 (CDK1), which cooperates
with cyclin B to promote mitotic processes. Liu et al. screened a kinome/phosphataseinhibitor library for compounds cytotoxic to RB1, PTEN and Trp53-deficient TNBC cells,
and identified a CDC25 inhibitor [83]. WEE1 is a nuclear kinase that phosphorylates CDK1
at the same site (Tyr15 ) as CDC25s [84]. The authors demonstrated that a WEE1 inhibitor
synergizes with a CDC25 inhibitor. How inhibitors of proteins with such opposing functions synergize is explained by their distinct roles during cell cycle progression [85]. WEE1
phosphorylates CDK1 during S phase at Thr14 and Tyr15 enabling a separate phosphorylation at Thr161 by CDK-activating kinase (CAK) during G2 phase [86]. This additional
phosphorylation at Thr161 allows CDC25s to fully activate CDK1 during M phase by executing dephosphorylation at Thr14 and Tyr15 [87]. In short, WEE1 and CDC25s cooperate
to promote the M phase progression in a sequential manner [88]. Other researchers demonstrated that the treatment of TNBC cells with a CDC25 inhibitor collaterally activates aresistant mechanism that is supported by AKT activity [89]. Thus, an PI3K inhibitor wouldsynergize with a CDC25 inhibitor. Although suppression of CDK1 activity was thought
to be a key mechanism of the action of these inhibitors, CDK1 antagonist did not fully
recapitulate it. This result indicates that there may be CDK1-indepdent functions of CDC25
and WEE1 in driving RB1-deficient cancers.
Prior to these reports, a study using Drosophila melanogaster system uncovered thatinactivation of rbf (the fly orthologue of RB1) and gig (the fly orthologue of tuberous sclerosis
complex 2 (TSC2)) synergistically induces cell death in imaginal disc cells placed under stressconditions [90]. TSC2 together with TSC1 inhibits the function of a complex containing
TORC1 and thereby suppresses tumor growth. Germline mutation in either of these genes
causes an autosomal dominant tumor-prone syndrome. The phenotypes observed in rbf-gig
double deficient fly cells appeared to depend on E2F transcription factors and the TOR(target of rapamycin) pathway. The synthetic lethal interaction between RB1 and TSC2 wasCancers 2021, 13, 3737

8 of 14also a case in multiple human prostate cancer cell lines. Inhibition of TSC2 in RB1-deficient
cancer cells increased oxidative stress in a superoxide dismutase 2 (SOD2)-dependent
manner, causing apoptosis [90]. The authors reasoned that this phenomenon was caused by
the suppression of de novo lipid synthesis [90]. A follow-up study done by the same groupindicated that dtsc1 (the fly orthologue of TSC1) also exhibited synthetic lethality wheninactivated together with rbf, which is associated with deregulated G1/S control, increased
DNA damage and energy stress [91]. These findings suggest that the mTOR pathway may
provide a vulnerable point in the treatment of RB1-deficient cancers in human.
An ‘ORFeome’ RNAi screen in Caenorhabditis elegans identified zfp-2, which encodesa zinc finger transcription factor, as a gene in a synthetic lethal relationship with lin-35(the C. elegans orthologue of RB1). These genes appeared to cooperate in somatic gonadal
development [92].
A study made use of multiple publicly available large-scale shRNA and siRNA screening data bases to explore genes in the synthetic lethal relationship with RB1 in TNBCs andnewly identified NUP88 and NUP214 nuclear pore components associated with the MAD2
spindle check protein. It further explored a kinase and bromodomain containing transcription factor called transcription initiation factor TFIID subunit 1 (TAF1) and rediscovered
SKP2 and SKP1 [93]. As above mentioned, MAD2 is overexpressed in RB1-deficient cells
and critically contributes to CIN [69]. TAF1 has been demonstrated to directly bind to RB1
to carry out basal transcription [94].
The products of most of genes so far identified due to their synthetic lethal relationshipwith RB1 are pharmacologically targetable (Table 1). Although loss of RB1 function is
generally associated with poor prognosis, it simultaneously gives rise to pharmacologicallytractable vulnerabilities leading tumor cells to carry lethal levels of DNA damage, CIN,
oxidative stress or E2F hyperactivation, causing cell death hopefully at a therapeuticallysatisfactory degree. As RB1 function is lost in a wide variety of cancer types at varied
stages, synthetic lethality screening in RB1-deficient cells needs to be extended to morevaries of cancers.
5. Targeting Loss of a Gene Neighboring RB1
The frequency that RB1 gene loci undergo homozygous deletion in cancer is the
fourth highest among tumor suppressor genes following INK4A, PTEN and SMAD4 [95].
Genetic deletion of SMAD4 that occurs in almost 30% of pancreatic ductal adenocarcinoma(PDAC) often associates simultaneous deletion of malic enzyme 2 (ME2) gene, as these genes
locate within 200 kb distance in the same chromosome. ME2 and its isozyme ME3 shareoxidative decarboxylase function to convert malate to pyruvate and to regenerate NADPH.
Depletion of ME3 in ME2-deficient pancreatic cancer cells induced lethal effects [96]. A
similar collateral vulnerability has been found in the glycolytic gene enolase 1 (ENO1) andENO2. ENO1 locates in the 1p36 locus which is frequently deleted in glioblastoma [97].
These studies demonstrated that targeting genes involved in genomic collapse in thechromosomal region surrounding tumor suppressor genes or frequently collapsed genome
regions may generate therapeutic benefits through collateral lethality.
Kohno et al. attempted a survey of metabolic genes located near to tumor suppressor genes that are frequently deleted in cancers. They then examined the frequency ofsimultaneous loss of such genes upon tumor suppressor gene loss in individual cancer
type. This effort discovered succinyl lyase A2 (SUCLA2) gene that locates around 302 kb
away from RB1 tumor suppressor gene in human chromosome 13q14.2. The germ line
mutation or deletion in SUCLA2 gene is causative of mitochondrial encephalomyopathy
and methylmalonic aciduria [98]. There is a case report of a patient who had undergone
a genomic deletion involving both SUCLA2 and RB1 gene. The patient developed encephalomyopathy and retinoblastoma simultaneously [99]. The simultaneous loss of RB1
and SUCLA2 was detected in various cancer types with varied prevalence. Surprisingly,
particularly in prostate cancers, SUCLA2 gene deletion was found to take place upon RB1
loss with near to 100% prevalence [100].

Cancers 2021, 13, 3737

Cancers 2021, 13, x

9 of 14

SUCLA2 contributes to a complex of enzymes that converts succinyl-CoA to succinate in the TCA cycle and generates GTP from GDP. Indeed, depletion of SUCLA2 inprostate cancer cells resulted in lower mitochondrial/respiratory activity and enhanced
glycolysis. These metabolic phenotypes continuously sustained as long as SUCLA2 wasdownregulated and thus were likely to be targeted aiming for treatment. Kohno et al.
sought the ways to make use of this finding. First, they attempted to additionally inhibit
SUCLG2 isozyme in the presence of RB1-SUCLA2 deletion, as it might completely disrupt
the conversion from succinyl-CoA to succinate. However, it did not kill such cells withsatisfactory efficacy [100].
Kohno et al. next screened a number of chemical libraries to find compounds that
selectively kill RB1-SUCLA2-deficeint prostate cancer cells [100]. This attempt hit several
compounds including 2-isopropyl-5-methylbenzo-1,4-quinone (thymoquinone), a naturalcompound which is known as one of major bioactive ingredients of Nigella Sativa [101](Figure 1). Although its anticancer and antioxidative functions were reported, the moleculartarget has been unidentified. In vivo treatment of immunodeficient mice xenografted with
RB1-SUCLA2-deficeint prostate cancer cells with thymoquinone resulted in a significantly
selective suppression of tumorous growth via apoptosis [100]. These findings indicatedthat the loss of a gene neighboring RB1 can be pharmacologically targeted in cancer therapy.
Homozygous deletion of RB1 is estimated to occur in more than 30% of advanced prostatecancer cases [11]. Heterozygous deletion of RB1 is also frequent in advanced prostate
10 of 15cancers, which is naturally associated with heterozygous loss of SUCLA2. Thymoquinone
appeared to be effective to SUCLA2-heterozyous prostate cancers as well [100]. Interestingly,
the Aurora A inhibitor that targets RB1 loss in a synthetic lethal manner synergizedpairs thymoquinone
functions of DREAM
complex,cooperatingwithRB1 losspromotetumor develwith
in prostatecancercells doubly
deficient
oftoRB1
and SUCLA2[100].
opment[104].
In
this
scenario,
insharp
contrast
to
SUCLA2,
loss
of
LATS2
generates
cerFurthermore, more varieties of cancers (bladder, ovarian, uterine, liver, cervical, esophageal,
tain
growthadvantages
toRB1-deficient
cancer.
It
isofinteresttoaddressif
pharmacobreast cancers, sarcoma, diffuse large B-cell lymphoma, etc.) are estimated to carry RB1logical enhancementin [100].
RB1-deficient
cancer is of
feasible.
As described
SUCLA2deletion withofupLATS2
to 5% function
prevalence
The development
drugs withstrongerabove,
BRCA2
gene
is
also
often
co-deleted
with
RB1
[74].
These
tumorsaregoodtherathymoquinone-like activity and identification of target molecule are awaited. Additionally,
peutictargetsof
PARP
inhibitors.
the discovery of biomarkers that help quick diagnosis of SUCLA2 deficiency in tumor
tissue would be forthcoming [100].

Figure 1. Thymoquinone targets SUCLA2 loss that collaterally takes place with RB1 homozygous or heterozygous deletion.
Figure 1. Thymoquinone targets SUCLA2 loss that collaterally takes place with RB1 homozygous or heterozygous deletion.

In addition to SUCLA2, there are some more genes neighboring RB1 locating even
6. Conclusions
closerto RB1. Lee et al. demonstrated that among such genes, loss of integral membraneprotein
2B (ITM2B),
chromosomal
condensation(CHC1L)and purinergic
receptor
P2Y
Inactivationof RB1functionsand the RB1-llike
pathway
is a majorhallmark ofcancer.
IrG
protein-coupledprotein of
5 (P2RY5)
functionmaycontributeto enhancereversible
dysfunction
RB1 often
predicts
poorprognosis
in manytumor
typespromotionof cancer.
inRB1-deficient
bladder cancer[102].
In a sharp
contrast,of SUCLA2
would
endow
However,the RB1-defecient
statusin cancers
mayturn outloss
to bea disguised
blessingforacancercertaindisadvantage
from
the
views
of
metabolism
andpharmacologicalvulnerability.
patients as it generates a variety of therapeutic benefits through the upregulation
Probably,
this explains
why RB1-SUCLA2
co-deletion
is not quiteprevalentin cancers
of RB1 targets,the presenceof genes which
are in a synthetic
lethalrelationshipwithother
RB1,
thanprostate cancer[100].
and collateralsynthetic
lethality due to SUCLA2 loss (Figure 2).

above, BRCA2 gene is also often co-deleted with RB1 [74]. These tumors are good therapeutic targets of PARP inhibitors.

Cancers 2021, 13, 3737

10 of 14

Dyson’s group carried out shRNA screenings to identify genes assisting RB1-induced
cellular senescence and discovered large tumor suppressor 2 (LATS2) [103]. Interestingly,
LATS2 locus are frequently deleted simultaneously with RB1 locus. LATS2 is primarily acomponent of Hippo pathway, but appeared to phosphorylate dual-specificity tyrosinephosphorylation-regulated kinase 1A (DYRK1A), which then phosphorylates LIN52 a
subunit of DREAM (DP-1, RB1, E2Fs and MuvB) complex. Therefore, loss of LATS2 impairs
functions of DREAM complex, cooperating with RB1 loss to promote tumor development [104]. In this scenario, in sharp contrast to SUCLA2, loss of LATS2 generates certain
growth advantages to RB1-deficient cancer. It is of interest to address if pharmacologicalenhancement of LATS2 function in RB1-deficient cancer is feasible. As described above,
Figure 1. Thymoquinone targetsBRCA2
SUCLA2geneloss thatcollaterally
takes place withheterozygous
deletion.
is alsooften co-deleted
withRB1RB1homozygous[74]. Theseortumors
are good
therapeutictargets of PARP inhibitors.
6. Conclusions
6.Inactivation
Conclusionsof RB1 functions and the RB pathway is a major hallmark of cancer. IrInactivation
of of
RB1
functionsand thepoor
RB pathway
a majorof cancer.
reversible
dysfunction
RB1
often predicts
prognosisisin
many hallmark
types of cancer.
Irreversible
dysfunction of
RB1inoften
predicts
poorout
prognosis
in many typesof cancer.
However,the RB1-defecient
statuscancers
may turn
to be a disguised
blessing
for
However,theRB1-defecient
statusincancers
may
turn
out
to
be
a
disguisedblessing
cancer patients as it generates a variety of therapeutic benefits through the upregulation forcancerpatients
it generates
a varietytherapeutic
benefits
throughthe upregulationof RB1targets,
the aspresenceof genes
whichofare
in a synthetic
lethalrelationshipwith RB1, ofRB1targets,
thepresence
of
genes
which
are
in
a
synthetic
lethalrelationshipwith RB1,and collateral synthetic lethality due to SUCLA2 loss (Figure 2).
and collateral synthetic lethality due to SUCLA2 loss (Figure 2).

Figure 2. The mechanisms which enable us to target RB1 loss in cancers. TME: tumor microenvironFigure 2. The mechanisms which enable us to target RB1 loss in cancers. TME: tumor microenvironment; CIN: chromosomal instability.
ment; CIN: chromosomal instability.
AuthorContributions:wrote
editedmanuscript.
other
AuthorContributions:
P.L.,P.L.,
S.K.,S.K.,
J.S. J.S.
andandC.T.C.T.
wrote
andandD.V.D.V.
editedthe themanuscript.
All Allotherauthors
provided
informationnecessaryfor preparing
manuscript. All authors have read andauthors
provided
informationnecessaryfor preparing
this thismanuscript.
agreed to the published version of the manuscript.
Funding: Japan Agency for Medical Research and Development, 21cm0106182h0006.
Funding: Japan Agency for Medical Research and Development, 21cm0106182h0006.
Acknowledgments: This study was supported by Funding Program for Next Generation WorldThis study
supported
byJapan,Funding
Program for
Generation
WorldAcknowledgments:
Leading
Researchers (LS049)
from wascabinetoffice ofGrant-in-Aid
forNext
Scientific
Research
Leading
Researchers(LS049)fromcabinetofficeofJapan,
Grant-in-Aid
for
Scientific
Research(17H03576, 17K19586, 19K22555 and 20H03509 to CT, and 17K14992 and 20K07612 to SK) from
(17H03576,
19K22555
and 20H03509
to CT, and 17K14992
and 20K07612
to SK) from MEXT,
MEXT,
Project17K19586,
for Cancer
Researchand Therapeutic(P-CREATE)(19cm0106164h0001,
Project for Cancer Research and Therapeutic (P-CREATE) (19cm0106164h0001, 20cm0106164h0001
and 21cm0106182h0006) from Japan Agency for Medical Research and Development (AMED), andTaNeDS (C1010568) and a collaborative research fund (C1010818) from Daiichi-Sankyo Co. Ltd.
Conflicts of Interest: The authors declare no conflict of interest.

References
1.
2.
3.
4.

Burkhart, D.L.; Sage, J. Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nat. Rev. Cancer 2008, 8, 671–682.
[CrossRef]
Harbour, J.W.; Dean, D.C. The Rb/E2F pathway: Expanding roles and emerging paradigms. Genes Dev. 2000, 14, 2393–2409.
[CrossRef] [PubMed]
Berry, J.L.; Polski, A.; Cavenee, W.K.; Dryja, T.P.; Murphree, A.L.; Gallie, B.L. The RB1 Story: Characterization and Cloning of theFirst Tumor Suppressor Gene. Genes 2019, 10, 879. [CrossRef]
Macpherson, D. Insights from mouse models into human retinoblastoma. Cell Div. 2008, 3, 9. [CrossRef]

Cancers 2021, 13, 3737

5.
6.
7.
8.

9.
10.
11.
12.

13.

14.

15.

16.

17.

18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.

11 of 14

Wikenheiser-Brokamp, K.A. Retinoblastoma family proteins: Insights gained through genetic manipulation of mice. Cell. Mol.
Life Sci. 2006, 63, 767–780. [CrossRef]
Wikenheiser-Brokamp, K.A. Retinoblastoma regulatory pathway in lung cancer. Curr. Mol. Med. 2006, 6, 783–793. [CrossRef]
Viatour, P.; Sage, J. Newly identified aspects of tumor suppression by RB. Dis. Model. Mech. 2011, 4, 581–585. [CrossRef]
Feugeas, O.; Guriec, N.; Babin-Boilletot, A.; Marcellin, L.; Simon, P.; Babin, S.; Thyss, A.; Hofman, P.; Terrier, P.; Kalifa, C.; et al.
Loss of heterozygosity of the RB gene is a poor prognostic factor in patients with osteosarcoma. J. Clin. Oncol. 1996, 14, 467–472.
[CrossRef] [PubMed]
Huang, H.J.; Yee, J.K.; Shew, J.Y.; Chen, P.L.; Bookstein, R.; Friedmann, T.; Lee, E.Y.; Lee, W.H. Suppression of the neoplastic
phenotype by replacement of the RB gene in human cancer cells. Science 1988, 242, 1563–1566. [CrossRef] [PubMed]
Ookawa, K.; Tsuchida, S.; Adachi, J.; Yokota, J. Differentiation induced by RB expression and apoptosis induced by p53 expression
in an osteosarcoma cell line. Oncogene 1997, 14, 1389–1396. [CrossRef]
Taylor, B.S.; Schultz, N.; Hieronymus, H.; Gopalan, A.; Xiao, Y.; Carver, B.S.; Arora, V.K.; Kaushik, P.; Cerami, E.; Reva, B.; et al.
Integrative genomic profiling of human prostate cancer. Cancer Cell 2010, 18, 11–22. [CrossRef]
Sharma, A.; Yeow, W.S.; Ertel, A.; Coleman, I.; Clegg, N.; Thangavel, C.; Morrissey, C.; Zhang, X.; Comstock, C.E.; Witkiewicz,
A.K.; et al. The retinoblastoma tumor suppressor controls androgen signaling and human prostate cancer progression. J. Clin.
Investig. 2010, 120, 4478–4492. [CrossRef] [PubMed]
Salah, M.; Nishimoto, Y.; Kohno, S.; Kondoh, A.; Kitajima, S.; Muranaka, H.; Nishiuchi, T.; Ibrahim, A.; Yoshida, A.; Takahashi, C.
An in vitro system to characterize prostate cancer progression identified signaling required for self-renewal. Mol. Carcinog. 2016,
55, 1974–1989. [CrossRef]
Kitajima, S.; Yoshida, A.; Kohno, S.; Li, F.; Suzuki, S.; Nagatani, N.; Nishimoto, Y.; Sasaki, N.; Muranaka, H.; Wan, Y.; et al. The
RB-IL-6 axis controls self-renewal and endocrine therapy resistance by fine-tuning mitochondrial activity. Oncogene 2017, 36,
5145–5157. [CrossRef]
Yoshida, A.; Kitajima, S.; Li, F.; Chaoyang, C.; Takegami, Y.; Kohno, S.; Wan, Y.; Hayashi, N.; Muranaka, H.; Nishimoto, Y.; et al.
MicroRNA-140 mediates RB tumor suppressor function to control stem cell-like activity through interleukin-6. Oncotarget 2017, 8,13872–13885. [CrossRef]
Li, F.; Kitajima, S.; Kohno, S.; Yoshida, A.; Tange, S.; Sasaki, S.; Okada, N.; Nishimoto, Y.; Muranaka, H.; Nagatani, N.; et al.
Retinoblastoma Inactivation Induces a Protumoral Microenvironment via Enhanced CCL2 Secretion. Cancer Res. 2019, 79,3903–3915. [CrossRef]
Kitajima, S.; Kohno, S.; Kondoh, A.; Sasaki, N.; Nishimoto, Y.; Li, F.; Mohammed, M.S.A.; Muranaka, H.; Nagatani, N.; Suzuki, M.;
et al. Undifferentiated State Induced by Rb-p53 Double Inactivation in Mouse Thyroid Neuroendocrine Cells and Embryonic
Fibroblasts. Stem Cells 2015, 33, 1657–1669. [CrossRef]
Kohno, S.; Kitajima, S.; Sasaki, N.; Takahashi, C. Retinoblastoma tumor suppressor functions shared by stem cell and cancer cellstrategies. World J. Stem Cells 2016, 8, 170–184. [CrossRef]
Calo, E.; Quintero-Estades, J.A.; Danielian, P.S.; Nedelcu, S.; Berman, S.D.; Lees, J.A. Rb regulates fate choice and lineage
commitment in vivo. Nature 2010, 466, 1110–1114. [CrossRef]
Bracken, A.P.; Ciro, M.; Cocito, A.; Helin, K. E2F target genes: Unraveling the biology. Trends Biochem. Sci. 2004, 29, 409–417.
[CrossRef] [PubMed]
Yamasaki, L.; Bronson, R.; Williams, B.O.; Dyson, N.J.; Harlow, E.; Jacks, T. Loss of E2F-1 reduces tumorigenesis and extends thelifespan of Rb1(+/−)mice. Nat. Genet. 1998, 18, 360–364. [CrossRef]
Fujiwara, K.; Yuwanita, I.; Hollern, D.P.; Andrechek, E.R. Prediction and genetic demonstration of a role for activator E2Fs in
Myc-induced tumors. Cancer Res. 2011, 71, 1924–1932. [CrossRef]
Rouaud, F.; Hamouda-Tekaya, N.; Cerezo, M.; Abbe, P.; Zangari, J.; Hofman, V.; Ohanna, M.; Mograbi, B.; El-Hachem, N.;
Benfodda, Z.; et al. E2F1 inhibition mediates cell death of metastatic melanoma. Cell Death Dis. 2018, 9, 527. [CrossRef]
Yao, H.; Lu, F.; Shao, Y. The E2F family as potential biomarkers and therapeutic targets in colon cancer. PeerJ 2020, 8, e8562.
[CrossRef]
Ma, Y.; Kurtyka, C.A.; Boyapalle, S.; Sung, S.S.; Lawrence, H.; Guida, W.; Cress, W.D. A small-molecule E2F inhibitor blocks
growth in a melanoma culture model. Cancer Res. 2008, 68, 6292–6299. [CrossRef]
Tadesse, S.; Caldon, E.C.; Tilley, W.; Wang, S. Cyclin-Dependent Kinase 2 Inhibitors in Cancer Therapy: An Update. J. Med. Chem.
2019, 62, 4233–4251. [CrossRef]
Sotillo, R.; Hernando, E.; Diaz-Rodriguez, E.; Teruya-Feldstein, J.; Cordon-Cardo, C.; Lowe, S.W.; Benezra, R. Mad2 overexpression
promotes aneuploidy and tumorigenesis in mice. Cancer Cell 2007, 11, 9–23. [CrossRef]
Schvartzman, J.M.; Duijf, P.H.; Sotillo, R.; Coker, C.; Benezra, R. Mad2 is a critical mediator of the chromosome instability
observed upon Rb and p53 pathway inhibition. Cancer Cell 2011, 19, 701–714. [CrossRef]
Perez, I.S. MAD2 in the Spotlight as a Cancer Therapy Regulator. Int. J. Cancer Oncol. 2016, 3, 1–8. [CrossRef]
Kastl, J.; Braun, J.; Prestel, A.; Moller, H.M.; Huhn, T.; Mayer, T.U. Mad2 Inhibitor-1 (M2I-1): A Small Molecule Protein-Protein
Interaction Inhibitor Targeting the Mitotic Spindle Assembly Checkpoint. ACS Chem. Biol. 2015, 10, 1661–1666. [CrossRef]
Li, J.; Dang, N.; Martinez-Lopez, N.; Jowsey, P.A.; Huang, D.; Lightowlers, R.N.; Gao, F.; Huang, J.Y. M2I-1 disrupts the in vivointeraction between CDC20 and MAD2 and increases the sensitivities of cancer cell lines to anti-mitotic drugs via MCL-1s. Cell
Div. 2019, 14, 5. [CrossRef]

Cancers 2021, 13, 3737

32.
33.
34.
35.
36.

37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.

50.
51.
52.
53.

54.
55.
56.
57.
58.
59.

12 of 14

Sage, J.; Straight, A.F. RB’s original CIN? Genes Dev. 2010, 24, 1329–1333. [CrossRef]
Amato, A.; Lentini, L.; Schillaci, T.; Iovino, F.; Di Leonardo, A. RNAi mediated acute depletion of retinoblastoma protein (pRb)promotes aneuploidy in human primary cells via micronuclei formation. BMC Cell Biol. 2009, 10, 79. [CrossRef]
Manning, A.L.; Longworth, M.S.; Dyson, N.J. Loss of pRB causes centromere dysfunction and chromosomal instability. Genes Dev.
2010, 24, 1364–1376. [CrossRef]
Knudsen, E.S.; Pruitt, S.C.; Hershberger, P.A.; Witkiewicz, A.K.; Goodrich, D.W. Cell Cycle and Beyond: Exploiting New RB1
Controlled Mechanisms for Cancer Therapy. Trends Cancer 2019, 5, 308–324. [CrossRef]
Shamma, A.; Takegami, Y.; Miki, T.; Kitajima, S.; Noda, M.; Obara, T.; Okamoto, T.; Takahashi, C. Rb Regulates DNA damage
response and cellular senescence through E2F-dependent suppression of N-ras isoprenylation. Cancer Cell 2009, 15, 255–269.
[CrossRef]
Kitajima, S.; Takahashi, C. Intersection of retinoblastoma tumor suppressor function, stem cells, metabolism, and inflammation.
Cancer Sci. 2017, 108, 1726–1731. [CrossRef]
Kitajima, S.; Li, F.; Takahashi, C. Tumor Milieu Controlled by RB Tumor Suppressor. Int. J. Mol. Sci. 2020, 21, 2450. [CrossRef]
Jin, X.; Ding, D.; Yan, Y.; Li, H.; Wang, B.; Ma, L.; Ye, Z.; Ma, T.; Wu, Q.; Rodrigues, D.N.; et al. Phosphorylated RB Promotes
Cancer Immunity by Inhibiting NF-kappaB Activation and PD-L1 Expression. Mol. Cell 2019, 73, 22–35.e26. [CrossRef]
Goel, S.; DeCristo, M.J.; Watt, A.C.; BrinJones, H.; Sceneay, J.; Li, B.B.; Khan, N.; Ubellacker, J.M.; Xie, S.; Metzger-Filho, O.; et al.
CDK4/6 inhibition triggers anti-tumour immunity. Nature 2017, 548, 471–475. [CrossRef]
Lu, X.; Horvitz, H.R. lin-35 and lin-53, two genes that antagonize a C. elegans Ras pathway, encode proteins similar to Rb and its
binding protein RbAp48. Cell 1998, 95, 981–991. [CrossRef]
Ceol, C.J.; Horvitz, H.R. dpl-1 DP and efl-1 E2F act with lin-35 Rb to antagonize Ras signaling in C. elegans vulval development.
Mol. Cell 2001, 7, 461–473. [CrossRef]
Lee, K.Y.; Ladha, M.H.; McMahon, C.; Ewen, M.E. The retinoblastoma protein is linked to the activation of Ras. Mol. Cell Biol.
1999, 19, 7724–7732. [CrossRef] [PubMed]
Takahashi, C.; Bronson, R.T.; Socolovsky, M.; Contreras, B.; Lee, K.Y.; Jacks, T.; Noda, M.; Kucherlapati, R.; Ewen, M.E. Rb andN-ras function together to control differentiation in the mouse. Mol. Cell Biol. 2003, 23, 5256–5268. [CrossRef]
Takahashi, C.; Contreras, B.; Bronson, R.T.; Loda, M.; Ewen, M.E. Genetic interaction between Rb and K-ras in the control ofdifferentiation and tumor suppression. Mol. Cell Biol. 2004, 24, 10406–10415. [CrossRef]
Takahashi, C.; Contreras, B.; Iwanaga, T.; Takegami, Y.; Bakker, A.; Bronson, R.T.; Noda, M.; Loda, M.; Hunt, J.L.; Ewen, M.E. Nras
loss induces metastatic conversion of Rb1-deficient neuroendocrine thyroid tumor. Nat. Genet. 2006, 38, 118–123. [CrossRef]
Takahashi, C.; Ewen, M.E. Genetic interaction between Rb and N-ras: Differentiation control and metastasis. Cancer Res. 2006, 66,
9345–9348. [CrossRef]
Takahashi, C.; Sasaki, N.; Kitajima, S. Twists in views on RB functions in cellular signaling, metabolism and stem cells. Cancer Sci.
2012, 103, 1182–1188. [CrossRef]
Muranaka, H.; Hayashi, A.; Minami, K.; Kitajima, S.; Kohno, S.; Nishimoto, Y.; Nagatani, N.; Suzuki, M.; Kulathunga, L.A.N.;
Sasaki, N.; et al. A distinct function of the retinoblastoma protein in the control of lipid composition identified by lipidomic
profiling. Oncogenesis 2017, 6, e350. [CrossRef]
Kulathunga, N.; Kohno, S.; Linn, P.; Nishimoto, Y.; Horike, S.I.; Zaraiskii, M.I.; Kumar, S.; Muranaka, H.; Takahashi, C. Peripubertal
high-fat diet promotes c-Myc stabilization in mammary gland epithelium. Cancer Sci. 2020, 111, 2336–2348. [CrossRef]
Zhang, J.; Benavente, C.A.; McEvoy, J.; Flores-Otero, J.; Ding, L.; Chen, X.; Ulyanov, A.; Wu, G.; Wilson, M.; Wang, J.; et al. A
novel retinoblastoma therapy from genomic and epigenetic analyses. Nature 2012, 481, 329–334. [CrossRef]
Zocchi, L.; Mehta, A.; Wu, S.C.; Wu, J.; Gu, Y.; Wang, J.; Suh, S.; Spitale, R.C.; Benavente, C.A. Chromatin remodeling protein
HELLS is critical for retinoblastoma tumor initiation and progression. Oncogenesis 2020, 9, 25. [CrossRef]
Benavente, C.A.; Finkelstein, D.; Johnson, D.A.; Marine, J.C.; Ashery-Padan, R.; Dyer, M.A. Chromatin remodelers HELLS andUHRF1 mediate the epigenetic deregulation of genes that drive retinoblastoma tumor progression. Oncotarget 2014, 5, 9594–9608.
[CrossRef]
Sanidas, I.; Morris, R.; Fella, K.A.; Rumde, P.H.; Boukhali, M.; Tai, E.C.; Ting, D.T.; Lawrence, M.S.; Haas, W.; Dyson, N.J. A Codeof Mono-phosphorylation Modulates the Function of RB. Mol. Cell 2019, 73, 985–1000.e6. [CrossRef] [PubMed]
DeCaprio, J.A. How the Rb tumor suppressor structure and function was revealed by the study of Adenovirus and SV40. Virology
2009, 384, 274–284. [CrossRef] [PubMed]
Christman, J.K. 5-Azacytidine and 5-aza-2’-deoxycytidine as inhibitors of DNA methylation: Mechanistic studies and theirimplications for cancer therapy. Oncogene 2002, 21, 5483–5495. [CrossRef]
Johnstone, R.W.; Licht, J.D. Histone deacetylase inhibitors in cancer therapy: Is transcription the primary target? Cancer Cell 2003,
4, 13–18. [CrossRef]
Lai, Y.S.; Chen, J.Y.; Tsai, H.J.; Chen, T.Y.; Hung, W.C. The SUV39H1 inhibitor chaetocin induces differentiation and shows
synergistic cytotoxicity with other epigenetic drugs in acute myeloid leukemia cells. Blood Cancer J. 2015, 5, e313. [CrossRef]
Huang, L.Y.; Lee, Y.S.; Huang, J.J.; Chang, C.C.; Chang, J.M.; Chuang, S.H.; Kao, K.J.; Tsai, Y.J.; Tsai, P.Y.; Liu, C.W.; et al.
Characterization of the biological activity of a potent small molecule Hec1 inhibitor TAI-1. J. Exp. Clin. Cancer Res. 2014, 33, 6.
[CrossRef]

Cancers 2021, 13, 3737

60.
61.
62.
63.

64.
65.
66.

67.

68.
69.
70.

71.

72.

73.
74.

75.

76.
77.
78.

79.
80.

81.

82.

83.

13 of 14Ji, P.; Jiang, H.; Rekhtman, K.; Bloom, J.; Ichetovkin, M.; Pagano, M.; Zhu, L. An Rb-Skp2-p27 pathway mediates acute cell cycle
inhibition by Rb and is retained in a partial-penetrance Rb mutant. Mol. Cell 2004, 16, 47–58. [CrossRef]
Lu, Z.; Bauzon, F.; Fu, H.; Cui, J.; Zhao, H.; Nakayama, K.; Nakayama, K.I.; Zhu, L. Skp2 suppresses apoptosis in Rb1-deficienttumours by limiting E2F1 activity. Nat. Commun. 2014, 5, 3463. [CrossRef]
Skapek, S.X.; Pan, Y.R.; Lee, E.Y. Regulation of cell lineage specification by the retinoblastoma tumor suppressor. Oncogene 2006,
25, 5268–5276. [CrossRef]
Kareta, M.S.; Gorges, L.L.; Hafeez, S.; Benayoun, B.A.; Marro, S.; Zmoos, A.F.; Cecchini, M.J.; Spacek, D.; Batista, L.F.; O’Brien, M.;
et al. Inhibition of pluripotency networks by the Rb tumor suppressor restricts reprogramming and tumorigenesis. Cell Stem Cell
2015, 16, 39–50. [CrossRef]
Kaelin, W.G., Jr. The concept of synthetic lethality in the context of anticancer therapy. Nat. Rev. Cancer 2005, 5, 689–698.
[CrossRef] [PubMed]
Lord, C.J.; Ashworth, A. PARP inhibitors: Synthetic lethality in the clinic. Science 2017, 355, 1152–1158. [CrossRef] [PubMed]
Gong, X.; Du, J.; Parsons, S.H.; Merzoug, F.F.; Webster, Y.; Iversen, P.W.; Chio, L.C.; Van Horn, R.D.; Lin, X.; Blosser, W.; et al.
Aurora A Kinase Inhibition Is Synthetic Lethal with Loss of the RB1 Tumor Suppressor Gene. Cancer Discov. 2019, 9, 248–263.
[CrossRef] [PubMed]
Oser, M.G.; Fonseca, R.; Chakraborty, A.A.; Brough, R.; Spektor, A.; Jennings, R.B.; Flaifel, A.; Novak, J.S.; Gulati, A.; Buss, E.;
et al. Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival. Cancer Discov. 2019,9, 230–247. [CrossRef] [PubMed]
Lyu, J.; Yang, E.J.; Zhang, B.; Wu, C.; Pardeshi, L.; Shi, C.; Mou, P.K.; Liu, Y.; Tan, K.; Shim, J.S. Synthetic lethality of RB1 andaurora A is driven by stathmin-mediated disruption of microtubule dynamics. Nat. Commun. 2020, 11, 5105. [CrossRef] [PubMed]
Manning, A.L.; Dyson, N.J. RB: Mitotic implications of a tumour suppressor. Nat. Rev. Cancer 2012, 12, 220–226. [CrossRef]
Shamma, A.; Suzuki, M.; Hayashi, N.; Kobayashi, M.; Sasaki, N.; Nishiuchi, T.; Doki, Y.; Okamoto, T.; Kohno, S.; Muranaka, H.;
et al. ATM mediates pRB function to control DNMT1 protein stability and DNA methylation. Mol. Cell Biol. 2013, 33, 3113–3124.
[CrossRef]
Velez-Cruz, R.; Manickavinayaham, S.; Biswas, A.K.; Clary, R.W.; Premkumar, T.; Cole, F.; Johnson, D.G. RB localizes to DNA
double-strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1. Genes
Dev. 2016, 30, 2500–2512. [CrossRef]
Marshall, A.E.; Roes, M.V.; Passos, D.T.; DeWeerd, M.C.; Chaikovsky, A.C.; Sage, J.; Howlett, C.J.; Dick, F.A. RB1 Deletion in
Retinoblastoma Protein Pathway-Disrupted Cells Results in DNA Damage and Cancer Progression. Mol. Cell Biol. 2019, 39.
[CrossRef]
Witkiewicz, A.K.; Chung, S.; Brough, R.; Vail, P.; Franco, J.; Lord, C.J.; Knudsen, E.S. Targeting the Vulnerability of RB Tumor
Suppressor Loss in Triple-Negative Breast Cancer. Cell Rep. 2018, 22, 1185–1199. [CrossRef] [PubMed]
Farago, A.F.; Yeap, B.Y.; Stanzione, M.; Hung, Y.P.; Heist, R.S.; Marcoux, J.P.; Zhong, J.; Rangachari, D.; Barbie, D.A.; Phat, S.; et al.
Combination Olaparib and Temozolomide in Relapsed Small-Cell Lung Cancer. Cancer Discov. 2019, 9, 1372–1387. [CrossRef][PubMed]Chakraborty, G.; Armenia, J.; Mazzu, Y.Z.; Nandakumar, S.; Stopsack, K.H.; Atiq, M.O.; Komura, K.; Jehane, L.; Hirani, R.;
Chadalavada, K.; et al. Significance of BRCA2 and RB1 Co-loss in Aggressive Prostate Cancer Progression. Clin. Cancer Res. 2020,
26, 2047–2064. [CrossRef] [PubMed]
Dick, F.A.; Goodrich, D.W.; Sage, J.; Dyson, N.J. Non-canonical functions of the RB protein in cancer. Nat. Rev. Cancer 2018, 18,
442–451. [CrossRef]
Zagorski, W.A.; Knudsen, E.S.; Reed, M.F. Retinoblastoma deficiency increases chemosensitivity in lung cancer. Cancer Res. 2007,
67, 8264–8273. [CrossRef]
Trere, D.; Brighenti, E.; Donati, G.; Ceccarelli, C.; Santini, D.; Taffurelli, M.; Montanaro, L.; Derenzini, M. High prevalence ofretinoblastoma protein loss in triple-negative breast cancers and its association with a good prognosis in patients treated withadjuvant chemotherapy. Ann. Oncol. 2009, 20, 1818–1823. [CrossRef] [PubMed]
Witkiewicz, A.K.; Ertel, A.; McFalls, J.; Valsecchi, M.E.; Schwartz, G.; Knudsen, E.S. RB-pathway disruption is associated withimproved response to neoadjuvant chemotherapy in breast cancer. Clin. Cancer Res. 2012, 18, 5110–5122. [CrossRef] [PubMed]
Robinson, T.J.; Liu, J.C.; Vizeacoumar, F.; Sun, T.; Maclean, N.; Egan, S.E.; Schimmer, A.D.; Datti, A.; Zacksenhaus, E. RB1 status intriple negative breast cancer cells dictates response to radiation treatment and selective therapeutic drugs. PLoS ONE 2013, 8,
e78641. [CrossRef]
Hijioka, S.; Hosoda, W.; Matsuo, K.; Ueno, M.; Furukawa, M.; Yoshitomi, H.; Kobayashi, N.; Ikeda, M.; Ito, T.; Nakamori, S.; et al.
Rb Loss and KRAS Mutation Are Predictors of the Response to Platinum-Based Chemotherapy in Pancreatic Neuroendocrine
Neoplasm with Grade 3: A Japanese Multicenter Pancreatic NEN-G3 Study. Clin. Cancer Res. 2017, 23, 4625–4632. [CrossRef]
Coussy, F.; El-Botty, R.; Chateau-Joubert, S.; Dahmani, A.; Montaudon, E.; Leboucher, S.; Morisset, L.; Painsec, P.; Sourd, L.;
Huguet, L.; et al. BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.
Sci. Transl. Med. 2020, 12. [CrossRef]
Liu, J.C.; Granieri, L.; Shrestha, M.; Wang, D.Y.; Vorobieva, I.; Rubie, E.A.; Jones, R.; Ju, Y.; Pellecchia, G.; Jiang, Z.; et al.
Identification of CDC25 as a Common Therapeutic Target for Triple-Negative Breast Cancer. Cell Rep. 2018, 23, 112–126.
[CrossRef] [PubMed]Cancers 2021, 13, 3737

14 of 14

Parker, L.L.; Piwnica-Worms, H. Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science 1992,
257, 1955–1957. [CrossRef]
85. Perry, J.A.; Kornbluth, S. Cdc25 and Wee1: Analogous opposites? Cell Div. 2007, 2, 12. [CrossRef]
86. Lolli, G.; Johnson, L.N. CAK-Cyclin-dependent Activating Kinase: A key kinase in cell cycle control and a target for drugs? Cell
Cycle 2005, 4, 572–577. [CrossRef]
87. Liu, K.; Zheng, M.; Lu, R.; Du, J.; Zhao, Q.; Li, Z.; Li, Y.; Zhang, S. The role of CDC25C in cell cycle regulation and clinical cancer
therapy: A systematic review. Cancer Cell Int. 2020, 20, 213. [CrossRef] [PubMed]
88. Lew, D.J.; Kornbluth, S. Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr. Opin. Cell Biol.
1996, 8, 795–804. [CrossRef]
89. Zacksenhaus, E.; Liu, J.C.; Granieri, L.; Vorobieva, I.; Wang, D.Y.; Ghanbari-Azarnier, R.; Li, H.; Ali, A.; Chung, P.E.D.; Ju, Y.;
et al. CDC25 as a common therapeutic target for triple-negative breast cancer—The challenges ahead. Mol. Cell Oncol. 2018, 5,e1481814. [CrossRef] [PubMed]
90. Li, B.; Gordon, G.M.; Du, C.H.; Xu, J.; Du, W. Specific killing of Rb mutant cancer cells by inactivating TSC2. Cancer Cell 2010, 17,
469–480. [CrossRef]
91. Gordon, G.M.; Zhang, T.; Zhao, J.; Du, W. Deregulated G1-S control and energy stress contribute to the synthetic-lethal interactions
between inactivation of RB and TSC1 or TSC2. J. Cell Sci. 2013, 126, 2004–2013. [CrossRef] [PubMed]
92. Ceron, J.; Rual, J.F.; Chandra, A.; Dupuy, D.; Vidal, M.; van den Heuvel, S. Large-scale RNAi screens identify novel genes thatinteract with the C. elegans retinoblastoma pathway as well as splicing-related components with synMuv B activity. BMC Dev.
Biol. 2007, 7, 30. [CrossRef]
93. Brough, R.; Gulati, A.; Haider, S.; Kumar, R.; Campbell, J.; Knudsen, E.; Pettitt, S.J.; Ryan, C.J.; Lord, C.J. Identification of highly
penetrant Rb-related synthetic lethal interactions in triple negative breast cancer. Oncogene 2018, 37, 5701–5718. [CrossRef]
94. Shao, Z.; Siegert, J.L.; Ruppert, S.; Robbins, P.D. Rb interacts with TAF(II)250/TFIID through multiple domains. Oncogene 1997,
15, 385–392. [CrossRef] [PubMed]
95. Bignell, G.R.; Greenman, C.D.; Davies, H.; Butler, A.P.; Edkins, S.; Andrews, J.M.; Buck, G.; Chen, L.; Beare, D.; Latimer, C.; et al.
Signatures of mutation and selection in the cancer genome. Nature 2010, 463, 893–898. [CrossRef] [PubMed]
96. Dey, P.; Baddour, J.; Muller, F.; Wu, C.C.; Wang, H.; Liao, W.T.; Lan, Z.; Chen, A.; Gutschner, T.; Kang, Y.; et al. Genomic deletionof malic enzyme 2 confers collateral lethality in pancreatic cancer. Nature 2017, 542, 119–123. [CrossRef] [PubMed]
97. Muller, F.L.; Colla, S.; Aquilanti, E.; Manzo, V.E.; Genovese, G.; Lee, J.; Eisenson, D.; Narurkar, R.; Deng, P.; Nezi, L.; et al.
Passenger deletions generate therapeutic vulnerabilities in cancer. Nature 2012, 488, 337–342. [CrossRef] [PubMed]
98. Carrozzo, R.; Dionisi-Vici, C.; Steuerwald, U.; Lucioli, S.; Deodato, F.; Di Giandomenico, S.; Bertini, E.; Franke, B.; Kluijtmans,
L.A.; Meschini, M.C.; et al. SUCLA2 mutations are associated with mild methylmalonic aciduria, Leigh-like encephalomyopathy,
dystonia and deafness. Brain 2007, 130, 862–874. [CrossRef] [PubMed]
99. Matilainen, S.; Isohanni, P.; Euro, L.; Lonnqvist, T.; Pihko, H.; Kivela, T.; Knuutila, S.; Suomalainen, A. Mitochondrial encephalomyopathy and retinoblastoma explained by compound heterozygosity of SUCLA2 point mutation and 13q14 deletion.
Eur. J. Hum. Genet. 2015, 23, 325–330. [CrossRef]
100. Kohno, S.; Linn, P.; Nagatani, N.; Watanabe, Y.; Kumar, S.; Soga, T.; Takahashi, C. Pharmacologically targetable vulnerability inprostate cancer carrying RB1-SUCLA2 deletion. Oncogene 2020, 39, 5690–5707. [CrossRef]
101. Khader, M.; Eckl, P.M. Thymoquinone: An emerging natural drug with a wide range of medical applications. Iran. J. Basic Med.
Sci. 2014, 17, 950–957.
102. Lee, S.; Jeong, J.; Majewski, T.; Scherer, S.E.; Kim, M.S.; Tuziak, T.; Tang, K.S.; Baggerly, K.; Grossman, H.B.; Zhou, J.H.; et al.
Forerunner genes contiguous to RB1 contribute to the development of in situ neoplasia. Proc. Natl. Acad. Sci. USA 2007, 104,
13732–13737. [CrossRef] [PubMed]
103. Tschop, K.; Conery, A.R.; Litovchick, L.; Decaprio, J.A.; Settleman, J.; Harlow, E.; Dyson, N. A kinase shRNA screen links LATS2
and the pRB tumor suppressor. Genes Dev. 2011, 25, 814–830. [CrossRef] [PubMed]
104. Tschop, K.; Dyson, N. Identifying players in the functional network around pRB. Cell Cycle 2011, 10, 3814–3815. [CrossRef][PubMed]84.