A WEE1 family business: Regulation of mitosis, cancer progression, and therapeutic target

Abstract

The inhibition of the DNA damage response (DDR) pathway in the treatment of cancer has recently gained interest, and different DDR inhibitors have been developed. Among them, the most promising ones target the WEE1 kinase family, which has a crucial role in cell cycle regulation and DNA damage identification and repair in both nonmalignant and cancer cells. This review recapitulates and discusses the most recent findings on the biological function of WEE1/PKMYT1 during the cell cycle and in the DNA damage repair, with a focus on their dual role as tumor suppressors in nonmalignant cells and pseudo-oncogenes in cancer cells. We here report the available data on the molecular and functional alterations of WEE1/PKMYT1 kinases in both hematological and solid tumors. Moreover, we summarize the preclinical information on 36 chemo/radiotherapy agents, and in particular their effect on cell cycle checkpoints and on the cellular WEE1/PKMYT1-dependent response. Finally, this review outlines the most important pre-clinical and clinical data available on the efficacy of WEE1/PKMYT1 inhibitors in monotherapy and in combination with chemo/radiotherapy agents or with other selective inhibitors currently used or under evaluation for the treatment of cancer patients.

Full text

R E V I E W Open Access
A WEE1 family business: regulation of
mitosis, cancer progression, and
therapeutic target
Andrea Ghelli Luserna di Rorà, Claudio Cerchione, Giovanni Martinelli and Giorgia Simonetti
*
Abstract
The inhibition of the DNA damage response (DDR) pathway in the treatment of cancer has recently gained interest,
and different DDR inhibitors have been developed. Among them, the most promising ones target the WEE1 kinase
family, which has a crucial role in cell cycle regulation and DNA damage identification and repair in both
nonmalignant and cancer cells. This review recapitulates and discusses the most recent findings on the biological
function of WEE1/PKMYT1 during the cell cycle and in the DNA damage repair, with a focus on their dual role as
tumor suppressors in nonmalignant cells and pseudo-oncogenes in cancer cells. We here report the available data
on the molecular and functional alterations of WEE1/PKMYT1 kinases in both hematological and solid tumors.
Moreover, we summarize the preclinical information on 36 chemo/radiotherapy agents, and in particular their effect
on cell cycle checkpoints and on the cellular WEE1/PKMYT1-dependent response. Finally, this review outlines the
most important pre-clinical and clinical data available on the efficacy of WEE1/PKMYT1 inhibitors in monotherapy
and in combination with chemo/radiotherapy agents or with other selective inhibitors currently used or under
evaluation for the treatment of cancer patients.
Keywords: WEE1 family kinases, WEE1, PKMYT1, Cell cycle, DNA repair, Pseudo-oncogene, Tumor suppressor
Background
The WEE1 kinase family consists of three serine/threo-
nine kinases sharing conserved molecular structures
and encoded by the following genes: WEE1 (WEE1 G2
Checkpoint Kinase), PKMYT1 (membrane-associated
tyrosine- and threonine-specific cdc2-inhibitory kinase),
and WEE1B (WEE2 oocyte meiosis inhibiting kinase). In
eukaryotic somatic cells, WEE1 and PKMYT1 play a key
role in cell cycle regulation and, in particular, they are
involved in the entry into mitosis [1]. Their role as regula-
tors is crucial during normal cell cycle progression and in
response to DNA damages, as part of the DNA damage
response (DDR) pathways. Similarly, WEE2 regulates cell
cycle progression and, in particular, meiosis [2]. Briefly,
WEE2 plays a dual regulatory role in oocyte meiosis by
preventing premature restart prior to ovulation and per-
mitting metaphase II exit at fertilization [3]. Despite the
identification of WEE2 somatic mutations (1.9% of cases)
and copy number (CN) alterations (22.5% of patients with
CN loss and 22.5% with CN gain) across several cancer
types (https://portal.gdc.cancer.gov), they have not been
functionally linked to tumor development so far. There-
fore, the following sections will be focused on WEE1 and
PKMYT1 kinases that have a well-recognized role in
oncology and hemato-oncology.
WEE1 and PKMYT1 in cell cycle regulation
WEE1 and PKMYT1 act as tumor suppressors in non-
malignant eukaryotic somatic cells. Similarly to other
DDR-related kinases, their main biological function is to
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* Correspondence: giorgia.simonetti@irst.emr.it
Biosciences Laboratory (Onco-hematology Unit), Istituto Scientifico
Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via P. Maroncelli
40, 47014 Meldola, FC, Italy
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https://doi.org/10.1186/s13045-020-00959-2
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prevent replication of cells with altered DNA. The main
downstream target of WEE1 family kinases is the
cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex,
also known as mitotic-promoting factor (MPF). WEE1
phosphorylates CDK1 on Tyr15 while PKMYT1 has a
dual activity on Tyr15 and Thr14 [4](Fig.1a). The
phosphorylation of those residues keeps the MPF
complex inhibited until the cell approaches mitosis.
WEE1 is located in the nucleus, while PKMYT1 is
associated with the endoplasmic reticulum and Golgi
apparatus [5,6], and regulates Golgi membrane
reassembly following mitosis [7]. Together, WEE1 and
PKMYT1 ensure that CDK1 remains inactive as it
shuttles into and out of the nucleus [8]. Through its
extra-nuclear localization, PKMYT1 can also promote
CDK1 cytosolic segregation. At the G2/M border, if no
DNA damage has been detected, CDK1 phosphorylation
on Tyr15 and Thr14 is rapidly removed by CDC25C phos-
phatase. In the nucleus, the CDK-activating kinase (CAK)
complex composed by cyclin-dependent kinase 7 (CDK7),
cyclin H1, and MAT1 promotes MPF complex activation
through the phosphorylation of CDK1(Thr161) [9,10].
The active MPF complex is then imported into the
nucleus through phosphorylation of cyclin B1 (Ser126,
Ser128, Ser133, and Ser147) [11]. This event is required to
enter mitosis. The relevance of WEE1 and PKMYT1 regu-
lation of CDK1 has been recently confirmed by in vivo
studies. Indeed, the replacement of the CDK1 inhibitory
phosphorylation sites with non-phosphorylatable amino
acids (CDK1
T14A/Y15F
) was embryonic lethal in mice [12].
Once activated, the MPF complex can phosphorylate
WEE1 and PKMYT1 to promote their inactivation via dif-
ferent cascades [5,13,14]. WEE1 is phosphorylated
(Ser123) by CDK1 at the onset of mitosis, thereby generat-
ing a binding motif for polo like kinase 1 (PLK1) and
casein kinase 2 (CK2), that in turn phosphorylate WEE1
(Ser53 and Ser121, respectively) [14,15]. Together, the
phosphorylation of the three Ser residues serves as a tag
for the degradation of WEE1 by the ubiquitin ligase SCFβ-
TrCP [13]. PKMYT1 is also phosphorylated by CDK1 and
PLK1 and this event promotes its degradation [16]. In
addition to the checkpoint function at the G2/M border,
recent findings highlighted a role of WEE1 in the regula-
tion of replication dynamics during S phase (intra S phase
checkpoint). When cells reach the S phase, replication is
initiated from a large number of replication origins trig-
gered through the activation of the pre-replication com-
plex [17] and following the activation of S phase specific
CDK, primarily CDK2 [18,19]. Similarly to CDK1, CDK2
regulation is controlled through Tyr15 phosphorylation
status, that is balanced by WEE1 (Fig. 1a) and cell division
cycle 25A (CDC25A) activity [20]. Both WEE1 and
CDC25A/C have been shown to modulate unperturbed
replication through regulating CDK1/CDK2 activity.
Monoallelic expression of CDK1
T14A/Y15F
induced replica-
tion stress and S phase arrest in mouse embryonic fibro-
blasts (MEFs), with substantial increase of γH2AX levels,
chromosomal fragmentation, and DDR activation, as a
consequence of intra-S phase DNA damage [12]. More-
over, unscheduled origin firing due to loss of WEE1 leads
to exhaustion of the replication protein A1 (RPA1) pool
and, as a consequence, to death during DNA replication
Fig. 1 WEE1 and PKMYT1 biological functions. aSchematic representation of WEE1 and PKMYT1 involvement in cell cycle checkpoints. WEE1
regulates the activity of both CDK1 and CDK2 kinases (trough phosphorylation of Tyr15) and is involved in the regulation of intra-S, G2/M, and M
phase cell cycle checkpoints. PKMYT1 selectively regulates CDK1 (through phosphorylation of Tyr15 and Thr14) and is plays a role in the G2/M
phase checkpoint. bSchematic representation of the regulation of MUS81-EME1/2 endonuclease complexes by WEE1 during S and G2/M cell
cycle phases. By inhibiting CDK2 or CDK1, WEE1 prevents MUS81 activation and the generation of DNA damages during S phase, and
chromosomes pulverization during G2/M phase
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(replication catastrophe). The intra S phase activity of
WEE1 is independent from PKMYT1 that is unable to
phosphorylate CDK2 [5]. In addition, WEE1, but not
PKMYT1, contributes to the control of mitosis exit.
Indeed, Wee1-deficient MEFs showed mitotic defects (e.g.,
in the number and position of centrosomes) that induce
arrest in mitosis or, in the majority of cells, mitotic slip-
page [21,22]. At the end of mitosis, WEE1 inhibits CDK1
through phosphorylation of its Tyr15 residue (Fig. 1a).
This event is dependent on the activation of the CTD
phosphatase subunit 1 (FCP1) that dephosphorylates and
activates WEE1 and other crucial component of the spin-
dle assembly checkpoint (SAC) complex [23]. Although
the precise mechanisms that regulate FCP1 activity is still
unknown, it has been showed that FCP1 promotes the
dephosphorylation of crucial SAC components, including
cell division cycle 20 (CDC20) and ubiquitin specific
peptidase 44 (USP44), thus promoting APC/C
Cdc20
activa-
tion and chromosome segregation [24–26]. Moreover,
WEE1 directly interacts with APC/C components, includ-
ing fizzy and cell division cycle 20 related 1 (CDH1),
CDC20, cell division cycle 27 (CDC27), and its deletion
enforced APC/C activity, resulting in alterations of the
level of APC/C substrates and mitosis progression at the
expense of genomic stability [21].
WEE1 regulates replication forks and genome
stability
The activity of WEE1 through the cell cycle can explain
its tumor suppressor function, at least in nonmalignant
cells. This observation was confirmed and disentangled
in preclinical studies. Indeed, conditional Wee1 hetero-
zygous deletion in the murine mammary epithelium
caused enhanced proliferation, with cells progressing
into mitosis while still undergoing DNA replication, and
consequent accumulation of DNA damage, resulting in
genomic instability and, ultimately, in tumor develop-
ment [21]. Biological processes such as DNA replication
and homologous recombination involve the formation of
branched DNA structures that physically link chromo-
somes. Such DNA structures needs to be disengaged
prior to entry into mitosis, in order to ensure proper
chromosome segregation. Eukaryotic cells evolved differ-
ent mechanisms to identify and process branched DNA
structures (Y-shape DNA) and the most important one
involves the structure-selective endonuclease MUS81.
MUS81 forms heterodimeric complexes with the non-
catalytic subunits EME1 or EME2 and recognizes Y-
shape DNA structures during DNA replication or during
mitosis (homologous recombination). The activity of
MUS81-EME1/2 complex is crucial to recover stalled
replication forks, during prolonged S phase arrest, and
to reset DNA junction between twin chromatids during
homologous recombination [27]. In unperturbed cells,
WEE1 protects replication forks and prevents the gener-
ation DNA damages and chromosome pulverization
through an indirect inhibition of MUS81 functionality
[28]. Indeed, WEE1 phosphorylates CDK1 and CDK2,
thus preventing the CDK-mediated phosphorylation and
activation of MUS81-EME1/2 complexes [29]. Lack of
WEE1-dependent regulation of MUS81-EME1/2 endonu-
cleases may lead to cleavage of unwanted DNA structure
(excessive replication forks), which would slow down
replication progression and increase genomic instability
[27,28](Fig.1b).
WEE1 and PKMYT1 deregulation in cancer cells
WEE1 and PKMYT1 act like oncogenes
The biological role of WEE1 and PKMYT1 in cancer
cells is not fully understood. Reduced WEE1 expression
has been detected in breast cancer compared with
normal tissues, independently of the tumor grade [21].
However, most findings suggest that both kinases act
like oncogenes rather than tumor suppressors. Indeed,
they are frequently overexpressed in both solid and
hematological tumors and a genome-wide CRISPR
screen of 563 cancer cell lines, showed that they are
essential for the cell viability of almost all cell lines [30].
The dependency of cancer cells on WEE1 family
proteins may be linked to the following mechanisms
(Fig. 2): (i) the high proliferation rate of cancer cells that
follows the activation of driver oncogenes (e.g. RAS,
MYC) needs to be sustained by a strong cell cycle regu-
lation machinery; (ii) cancer cells frequently inactivate
p53, which is a key gatekeeper of G0/G1 and S phases
and, as a consequence, the regulation of cell cycle is
sustained entirely by the G2/M checkpoint; (iii) the
over-expression of DDR-related kinases is fundamental
to maintain a tolerable level of genetic instability, an
intrinsic feature of cancer cells [31,32]. Therefore, we
can speculate that, once the malignant transformation
process has been induced, WEE1 upregulation exerts a
pro-tumorigenic functions by securing a tolerable level
of genomic instability to cancer cells. The following
sections summarize the current knowledge on the mo-
lecular and functional alterations of WEE1 and PKMY
T1 in hematological and solid tumors.
WEE1 and PKMYT1 genetic lesions in cancer
WEE1 and PKMYT1 are rarely mutated in cancer
patients, with an overall mutation frequency of 1.2% and
0.2%, respectively (https://portal.gdc.cancer.gov). The
distribution of somatic mutations is highly heteroge-
neous across cancer types (WEE1: 0.2–7.6%; PKMYT1:
0.1–3.6%), with a higher frequency in uterine corpus
endometrial carcinoma (UCEC) and tumors of the
gastrointestinal tract (stomach and colon adenocarcin-
oma, Fig. 3a, b). In particular, WEE1 mutations have
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been reported in 7.6% of UCEC cases. Moreover, PKMY
T1 lesions have been also detected in 2.7% of diffuse
large B cell lymphoma (DLBC). Conversely, both kinase
genes are rarely mutated in brain lower grade glioma
(LGG), ovarian serous cystadenocarcinoma (OV), pros-
tate adenocarcinoma (PRAD), and sarcoma (SARC), with
a frequency lower than 0.5%. Both genes are mainly tar-
geted by missense mutations that preferentially cluster
in the region encoding the WEE1 kinase domain and its
surroundings (Fig. 3c), suggesting a potential gain of
function effect of the kinase activity. Conversely, the
mutations are scattered throughout the PKMYT1 se-
quence (Fig. 3d). Little is known about the functional
consequences of WEE1 and PKMYT1 mutations. In the
majority of cancer types, the transcript expression in the
mutated cases is higher than the median value of the
entire cohort (https://www.cbioportal.org), supporting
once more an oncogenic function. In pancreatic adeno-
carcinoma (PA) patients and cell lines, an insertion was
identified in the WEE1 poly-T track, which contains the
binding site of the HuR RNA binding protein [33]. The
insertion resulted in decreased WEE1 expression upon
mitomycin-induced DNA damage, which would argue
against a protective effect of the mutation. Copy number
alterations (CNAs) represent a more frequent event
compared with mutations, with the WEE1 gene being
predominantly involved in CN loss (23.7% of cases
versus 7.8% of patients with CN gains), while PKMYT1
showing a higher percentage of CN gain (15.9% versus
12.0% of CN loss, Fig. 3e, f). The predominance of
WEE1 deletion events (6.3% versus 3.25% of cases with
amplification) was also observed in breast cancer, in line
with its reduced expression, as mentioned above [21].
Overall, cancer types showing the highest recurrence (>
10%) of CNAs were OV (27.7%), lung squamous cell
carcinoma (LUSC, 14.8%), uterine carcinosarcoma (UCS,
12.5%), and SARC (11.2%) for WEE1 and OV (18.8%),
bladder urothelial carcinoma (BLCA, 13.7%), and
esophageal carcinoma (ESCA, 10.3%) for PKMYT1.Of
note, OV and LUSC have been classified as tumors with
multiple recurrent chromosomal gains and losses [34],
which may suggest a bystander effect related to chromo-
somal instability in these tumor types, especially in the
case of WEE1 deletion, that is unexpected, based on the
general oncogenic function exerted by the kinase.
WEE1 and PKMYT1 functional role in hematological and
solid tumors
Few studies have analyzed WEE1 and PKMYT1 expres-
sion in hematological malignancies. Our group showed
that WEE1 kinase is highly expressed in acute lympho-
blastic leukemia (ALL) cell lines and primary cells in
comparison with normal hematopoietic cells, and that
PKMYT1 is upregulated in relapsed ALL samples
compared with nonmalignant hematopoietic cells [35].
Moreover, we demonstrated that ALL cells are
dependent on WEE1 functionality for their survival and
proliferation and that PKMYT1 levels may influence the
sensitivity to the WEE1 inhibitor AZD-1775 [35]. Similar
results on the role of WEE1 were obtained in multiple
myeloma (MM), acute myeloid leukemia (AML), chronic
myeloid leukemia (CML), and chronic lymphocyte
leukemia (CLL) [36–39]. In AML cells, WEE1 and
PKMYT1 are key gene discriminating between FLT3-
ITD, FLT3-TKD, and NRAS-mutated samples. They
were expressed at lower levels selectively in FLT3-ITD
specimens in comparison with wild-type cells, suggesting
either a tumor suppressor role in the leukemogenic
process or a potential vulnerability n this AML subtype
[40]. Pharmacological WEE1 inhibition alone or in com-
bination with histone deacetylase inhibitors showed
therapeutic potential in FLT3-ITD AML, confirming
their dependency on WEE1 activity [41]. Since FLT3-
ITD AML have intrinsic homologous recombination
repair defects [42]. WEE1 inhibition may exacerbate the
cell genotoxic stress by disrupting multiple cell cycle
checkpoints. WEE1 has been showed to be a valuable
target also for lymphoma patients [43]. In parallel,
Fig. 2 WEE1 family proteins role as tumor suppressors or pseudo-oncogenes in non-malignant and cancer cells
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PKMYT1 proved to be essential for MM cell line viabil-
ity, since its downregulation strongly decreased cell
growth, while inducing apoptosis [44].
WEE1 and PKMYT1 are also over-expressed in solid
tumors, including hepatocellular carcinoma, colon cancer,
glioblastoma, non-small-cell lung cancer (NSCLS), neuro-
blastoma, and gastric cancers [31,32,45–47]. High WEE1
expression has been associated with negative prognostic
factors including lymph node involvement, induction of
metastasis, increased biomarkers of proliferation (CCND1,
Ki67, or CCNA1) and resistance to treatments (radiother-
apy or chemotherapy) [48–51]. Elevated PKMYT1 levels
have been associated with tumor progression, a more
aggressive disease, the induction of metastasis at least in
NSCLS patients [45] and, generally, with poor prognosis.
Depending on the cancer subtype, the expression of
WEE1 and PKMYT1 has been linked with the activation
of cellular pathways crucial for the specific disease. In
melanoma cells, WEE1 silencing caused an increase of
phospho p38 protein levels, indicating a role in the
regulation of p38/MAPK pathway activation during p53-
independent DNA damage response [49]. In hepatocellu-
lar carcinoma and colorectal cancers, PKMYT1 regulates
epithelial-mesenchymal transition (EMT), a process rele-
vant to tumor progression, invasion, metastasis, and drug
resistance, through the activation of the beta-catenin/TCF
Fig. 3 WEE1 and PKMYT1 mutations and copy number alterations (CNAs) in cancer. aFrequency of patients with WEE1 or bPKMYT1 gene
mutations across cancers from TCGA cohorts. cDistribution of mutations according to the WEE1 and dPKMYT1 amino acid (aa) sequence and
protein domains (WEE1 transcript ENST00000450114, 646 aa; PKMYT1 transcript ENST00000262300, 499 aa). eFrequency of patients with copy
number gain or loss in WEE1 or fPKMYT1 across cancers (https://portal.gdc.cancer.gov;ACC adrenocortical carcinoma, BLCA bladder urothelial
carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, COAD colon
adenocarcinoma, CHOL cholangiocarcinoma, DLBC diffuse large B cell lymphoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme,
HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRK kidney renal clear cell carcinoma, KIRP kidney renal papillary cell
carcinoma, LGG brain lower grade glioma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma,
MESO mesothelioma, OV ovarian serous cystadenocarcinoma, PAAD pancreatic adenocarcinoma, PCPG pheochromocytoma and paraganglioma,
PRAD prostate adenocarcinoma, READ rectum adenocarcinoma, SARC sarcoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma,
TGCT testicular germ cell tumors, THYM thymoma, UCS uterine carcinosarcoma, UCEC uterine corpus endometrial carcinoma)
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signaling [32,46], while PKMYT1 has been reported to
control Notch pathway in NSCLC [45]. In particular,
crucial component of the pathway, including NOTCH1,
p21, and HES1 are downregulated by chemical inhibition
of PKMYT1 [45]. In neuroblastic tumors, PKMYT1 is
required to stabilize MYCN protein, which is a crucial
proto-oncogene for this cancer types [52]. Moreover, in
esophageal squamous cell carcinoma (ESCC) cell lines and
primary cells, the expression of PKMYT1 is associated
with and regulates the activation of the AKY/mTOR path-
way [53](Table1). Taken together, this evidence suggests
a broad role of WEE1/PKMYT1 besides the DNA damage
response pathway that may increase the interest towards
its therapeutic targeting.
Development of WEE1 and PKMYT1 inhibitors
WEE1 and PKMYT1 inhibitors have single agent and
chemo-sensitizer effects
Due to their potential oncogenic role, WEE1 and PKMY
T1 have been investigated as therapeutic targets for
hematological and solid tumors. Several pharmacological
inhibitors have been designed and subsequently vali-
dated in different cancer models. The available literature
highlights a common mechanism of action of WEE1/
PKMYT1 inhibitors in cancer cells either in single agent
or in combination with DNA damaging agents (chemo-
therapy/radiotherapy). WEE1/PKMYT1 kinase inhibition
causes G2/M cell cycle checkpoint override, premature
mitotic entry, and cell death during mitosis, through a
mechanism generally known as mitotic catastrophe
(Fig. 4a). From a biological point of view, the inhibition
of WEE1 kinase causes a significant reduction of phospho-
CDK1 (Tyr15), thus promoting the accumulation of active
CDK1-cyclin B1 complex and, consequently, mitotic entry.
The beginning of mitosis is also associated with a progres-
sive accumulation of DNA damages and the degeneration
in mitotic catastrophe. The sensitivity to WEE1 kinase
inhibitors in relation to TP53 mutational status remains
controversial. Indeed, some studies reported increased sen-
sitivity of TP53 mutant cell lines to WEE1 inhibitors in
comparison to TP53 wild-type ones [62,63], while others
showed no association between p53 functionality and the
effectiveness of WEE1 inhibition [35,64]. These discrepan-
cies may be linked to the intrinsic chromosomal instability
of the analyzed tumors and to additional alterations deregu-
lating the G1 checkpoint in TP53 wild-type cases that may
enhance the sensitivity to WEE1 targeting.
Regarding the role of WEE1 inhibitors as chemo-
sensitizer agents, a large number of studies demonstrated
a synergistic activity between DNA damaging agents
(chemotherapy including doxorubicin, cytarabine, metho-
trexate, cisplatin, clofarabine, etoposide, 5-fluorouracil,
and radiotherapy) and different WEE1/PKMYT1 inhibi-
tors in preclinical models [48,56,65–69]. The mechanism
of action of the combination is based on the inhibition of
the DDR pathway following induction of DNA damage in-
duced by the chemotherapy or radiotherapy agents. In this
scenario, cancer cells with damaged DNA fail to arrest cell
cycle, continue to proliferate, and accumulate massive
DNA damage until a point of no return (Fig. 4b). Indeed,
several DNA damaging agent promote the indirect activa-
tion of WEE1 and PKMYT1 kinases, as showed mostly by
the activation of cell cycle checkpoints (S and G2/M
checkpoints) in cancer cells. We summarized in Table 2
the results of preclinical studies in which the effect of
different chemotherapy agents or radiotherapy has been
evaluated in terms of cell cycle perturbation and altered
expression of WEE1 or PKMYT1 following in vitro or
Table 1 WEE1 and PKMYT1 molecular alterations in hematological and solid tumors according to literature
Gene Genetic alteration Disease Effect/prognostic value Reference
Hematological tumors
WEE1 Over-expression ALL; AML; MM; CML;
CLL; DLBCL
Crucial for cell viability of cancer cells (experimentally proven). [35–40,43,54]
Copy number Gain AML Biological effect or prognostic value unknown [55]
PKMYT1 Over-expression ALL; MM Crucial for cell viability of cancer cells (experimentally proven). [35,44]
Solid tumors
WEE1 Over-expression GC; MaM; GL; OC; CC Associated with lymph node involvement, induction of
metastasis, increased biomarkers of proliferation (CCND1, Ki67
or CCNA1), resistance to treatment and poor overall survival.
[48–51,56–60]
Mutation PA Insertion causing decrease WEE1 expression upon DNA
damage
[33]
PKMYT1 Over-expression HC; CC; GLB; NSCLC; N; GS Associated with tumor progression, aggressive disease and
poor overall survival.
[31,32,45–47]
Mutation N Biological effect or prognostic value unknown [61]
ALL acute lymphoblastic leukemia, AML acute myeloid leukemia, MM multiple myeloma, CML chronic myeloid leukemia, CLL chronic lymphocyte leukemia, DLBCL
diffuse large B cell lymphoma, GC gastric cancer, MaM malignant melanoma, GL gliomas, OC ovarian cancer, CC colorectal cancer, PA pancreatic adenocarcinoma,
HC hepatocellular carcinoma, GLB glioblastoma, NSCLC non-small-cell lung cancer, Nneuroblastoma
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in vivo treatment. Taken together, the abovementioned
data prove that WEE1 and PKYMYT1 are ideal targets to
override cell cycle checkpoint regulation and to improve
the efficacy of DNA-damaging agents. In particular,
tumors with a high level of chromosomal instability may
respond to WEE1/PKMYT1 inhibition per se, while cases
with a more stable genomic asset may benefit of the
combination between DNA-damaging agents and WEE1
family kinase inhibitors. The following sections reports
the main preclinical and clinical findings obtained using
small molecules inhibitors of WEE1 and PKMYT1 kinases.
Preclinical studies of WEE1 and PKMYT1 inhibitors
Several targeted compounds showed an inhibitory activ-
ity on WEE1 and PKMYT1 kinases and their efficacy
was proven in a number of tumor types. Table 3shows
the main preclinical studies that used WEE1/PKMYT1
inhibitors in single agent or in combination with chemo/
radiotherapy agents in different tumor types.
PD0166285 is the first reported drug, with an inhibi-
tory activity against WEE1, PKMYT1, and a range of
other kinases including c-Src, EGFR, FGFR1, CHK1, and
PDGFRb [151].
Adavosertib (AZD-1775) is the first highly potent and
selective WEE1 inhibitor. A large number of preclinical
studies evaluated its efficacy in single agent and in
combinatory approaches. Regarding the mechanism of
action, adavosertib induces S and/or G2/M cell cycle
checkpoints override, depending on cancer types, when
used in monotherapy. Cell cycle perturbation is associ-
ated with a progressive accumulation of DNA damages
and by the induction of apoptosis [35,99,119–122].
This last event is cell cycle phase-dependent and can
occur (i) as a consequence of S phase checkpoint
Fig. 4 Mechanism of action of WEE1/PKMYT1 inhibitors for the treatment of cancer cells. aSchematic representation of WEE1/PKMYT1 inhibition
as monotherapy. In cancer cells, oncogenes promote high rate of proliferation, replication stress and the over-expression of WEE1/PKMYT1
kinases. In this scenario, cancer cells need WEE1 and PKMYT1 to sustain replication stress and proliferation. The inhibition of WEE1/PKMYT1 results
in the accumulation of DNA damages, the increase of genetic instability and induction of apoptosis. bSchematic representation of WEE1/PKMYT1
inhibition in combination with DNA damaging agents. Cancer cells respond to DNA damages by activating WEE1/PKMYT1 kinases. The inhibition
of WEE1/PKMYT1 enhances the cytotoxicity of DNA damaging agents by inhibiting DNA repair and promoting cell cycle progression even in the
presence of DNA damages. Therefore, cancer cells accumulate massive DNA damages until a point of no return
Ghelli Luserna di Rorà et al. Journal of Hematology & Oncology (2020) 13:126 Page 7 of 17
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override, when cancer cells start DNA replication even
in the presence of DNA damages (replicative catastro-
phe); (ii) following G2/M phase checkpoint override,
that results in forced entry into mitosis, even in the pres-
ence of DNA damages (mitotic catastrophe).
In combination strategies, adavosertib was able to
enhance the cytotoxicity of chemo/radiotherapy agents,
by inducing cell cycle checkpoint override, inhibition of
DNA damage repair, and induction of apoptosis [35,37,
38,92,121,127–129]. The chemo-sensitizer efficacy of
DDR inhibitors has been linked to drug scheduling [94,
152,153]. Recently in pancreatic adenocarcinoma cells,
it has been reported that the efficacy of a triple regimen
combining gemcitabine, CHK1, and WEE1 inhibitors is
strictly dependent on the timing of drug administration.
Indeed, the maximum effect of the combination is
Table 2 Effects of standard of care chemo/radiotherapy agents on cell cycle checkpoints activation
Chemotherapy agents/radiotherapy Intra S checkpoint G2/M checkpoint WEE1 and/or PKMYT1 experimentally
proven involvement in cancer model
Actinomycin No Yes [70,71] WEE1 upregulation [71]
Azacitidine No Yes [72]NA
Bleomycin No Yes [73]NA
Carboplatin No Yes [74]NA
Cisplatin No Yes [75,76] WEE1 inhibition enhanced cytotoxicity [76,77]
Cyclophosphamide NA NA WEE1 upregulation [78]
Cytarabine Yes [79,80] Yes [79,80] WEE1 upregulation [38,81]
Clofarabine Yes [35] No WEE1 inhibition enhanced cytotoxicity [35]
Daunorubicin Yes [82] Yes [82]NA
Decitabine No Yes [83]NA
Docetaxel No Yes [84,85]NA
Doxorubicin No Yes [86] WEE1 upregulation [86]; WEE1 inhibition
enhanced cytotoxicity [35]
Epirubicin No Yes [87,88] WEE1 inhibition enhanced cytotoxicity [89]
Epothilone No Yes [90]NA
Etoposide No Yes [91] WEE1 inhibition enhanced cytotoxicity [67]
Fluorouracil Yes [92] No WEE1 inhibition enhanced cytotoxicity [92]
Fludarabine Yes [80]No NA
Gemcitabine Yes [80] No WEE1 upregulation [93]; WEE1 inhibition
enhanced cytotoxicity [94]
Hydroxyurea Yes [95] No WEE1 inhibition enhanced cytotoxicity [96]
Idarubicin No Yes [97]NA
Irinotecan No Yes [98] WEE1 inhibition enhanced cytotoxicity [99]
Mechlorethamine Yes [100] Yes [100]NA
Mercaptopurine Yes [101]No NA
Methotrexate Yes [102] No WEE1 inhibition enhanced cytotoxicity [103]
Mitoxantrone No Yes [104] WEE1 inhibition enhanced cytotoxicity [78]
Oxaliplatin No Yes [105] WEE1 inhibition enhanced cytotoxicity [106]
Paclitaxel No Yes [107] WEE1 inhibition enhanced cytotoxicity [108]
Pemetrexed Yes [109] No WEE1 inhibition enhanced cytotoxicity [110]
Radiotherapy (ionizing radiation) No Yes [111] WEE1 inhibition enhanced cytotoxicity [68]
Teniposide Yes [112] Yes [112]NA
Thioguanine Yes [113] Yes [113] WEE1 inhibition enhanced cytotoxicity [65]
Topotecan No Yes [114] WEE1 inhibition enhanced cytotoxicity [115]
Vinblastine No Yes [116]NA
Vincristine No Yes [117] WEE1 inhibition enhanced cytotoxicity [118]
Ghelli Luserna di Rorà et al. Journal of Hematology & Oncology (2020) 13:126 Page 8 of 17
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obtained when gemcitabine and CHK1 inhibitors are
administered simultaneously (thus inducing replicative
stress) and adavosertib is added at a later time [94].
Moreover, strong synergism has been observed by
combining adavosertib with small molecules, including
DDR-related inhibitors (CDK2 [89], CDK4-6 [149],
Table 3 Preclinical studies evaluating the effect of WEE1 inhibitors in monotherapy or in combination with chemotherapy/
radiotherapy in cancer
Inhibitor Treatment Cancer model Main biological effect References
PD0166285 M GBM-astrocytoma -G2/M checkpoint override
-Forced mitotic entry
[51]
Adavosertib M MM, ALL, AML TNBC, DLBCL, MCL -G2/M checkpoint override
-Forced mitotic entry
-Mitotic catastrophe
-Replicative catastrophe
[35,99,119–122]
PD0166285 +R GBM-astrocytoma -Mitotic catastrophe
-Inhibition of DNA repair
[51]
Adavosertib +R CC, LC, BC, PC, OC, DLBCL, ES -Increased DNA damage
-Induction of apoptosis
-Mitotic catastrophe
[78,99,123–126]
Adavosertib +C AML, ALL, MM, BC, CC, GC, DLBCL -S or G2/M checkpoint override
-Increased DNA damaged
-Induction of apoptosis
[35,37,38,76,92,99,
121,127–129]
Adavosertib +HDAC i AML, HNSCC -Replication stress
-Replicative catastrophe
-Increased DNA damage
-Inhibition of DNA repair
[41,130,131]
Adavosertib +ATR i AML, DLBCL, MCL, BC -Replication stress
-Replicative catastrophe
-Increased DNA damaged
-Inhibition of DNA repair
[132–135]
Adavosertib +mTOR i AML, ALL, OC, NSCLC -Inhibition of DNA repair [136–139]
Adavosertib +CHK1 i MCL, DLBCL, ALL, AML -Replication stress
-Increased DNA damage
-Replicative catastrophe
[103,140–142]
Adavosertib +BCL2i/MCL-1 i DLBCL -Force mitotic entry
-Increase DNA damage
-INDUCTION of apoptosis
[143]
Adavosertib +PARP1 i NSCLC, AML, ALL -G2/M checkpoint override
-Replication stress
-Increased DNA damage
-Inhibition of DNA repair
[126,144–146]
Adavosertib +AURORA A i HNSCC -Forced mitotic entry
-Mitotic catastrophe
[147]
Adavosertib +CDK2 i BC -Replication stress
-Replicative catastrophe
[89]
Adavosertib +SIRT1 i LC -Inhibition of DNA repair [148]
Adavosertib +CDK4-6 i S -Replication stress [149]
Adavosertib +BCR-ABL1 i ALL -Inhibition of DNA repair -G2/M
checkpoint override
[35]
Adavosertib +Proteasome i MM -G2/M checkpoint override
-Forced mitotic entry
-Inhibition of DNA repair
[36]
Adavosertib +BET i NSCLC -Inhibition of DNA repair
-Forced mitotic entry
-Mitotic catastrophe
[150]
Mmonotherapy, Rradiotherapy, Cchemotherapy, ALL acute lymphoblastic leukemia, AML acute myeloid leukemia, MM multiple myeloma, DLBCL diffuse large B
cell lymphoma, MCL mantle cell lymphoma, GC gastric cancer, GL gliomas, OC ovarian cancer, CC colorectal cancer, PC pancreatic cancer, ES esophageal cancer, HC
hepatocellular carcinoma, GLB glioblastoma, NSCLC non-small-cell lung cancer, Nneuroblastoma, Ssarcomas, LC lung cancer, BC breast cancer, HNSCC head and
neck squamous cell carcinoma, TNBC triple negative breast cancer
Ghelli Luserna di Rorà et al. Journal of Hematology & Oncology (2020) 13:126 Page 9 of 17
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CHK1 [103,140–142], ATM [132–135], AURORA A
[147], PARP1 [144], SIRT1 [148] inhibitors), histone
deacetylase (HDAC) inhibitors [41,130,131], tyrosine
kinase inhibitors (BCR-ABL1 inhibitors [35]), anti-
apoptotic protein inhibitors (BCL2 and MCL1 inhibitors
[143]), mTOR inhibitor [136–139], and proteasome in-
hibitors [36].
We have recently reported synergistic effects of adavo-
sertib in combination with different tyrosine kinase
inhibitors in both BCR-ABL1-positive and -negative
ALL cell lines and primary cells. Interestingly, strong
synergism was found in BCR-ABL1-negative ALL cell
lines treated with adavosertib in combination with bosu-
tinib isomer. In the study, we speculated that the strong
cytotoxic effect of the combination was due to the con-
comitant inhibition of WEE1 and PKMYT1 kinases [35].
Indeed, no selective inhibitor has been currently devel-
oped to target its functionality. However, several known
tyrosine kinase inhibitors have an inhibitory off-target
effect on PKMYT1. Among them, compounds com-
monly used for the treatment of BCR-ABL1-positive
CML and ALL, as dasatinib and bosutinib (and a struc-
tural isomer of bosutinib [154,155]) were shown to
inhibit PKMYT1 activity.
Overall, the data suggest that WEE1/PKMYT1 inhib-
ition is a suitable pharmacological target for combin-
ation strategies in cancer. The broad spectrum of
activities exerted by the two kinases, and especially by
WEE1, across the cell cycle, makes them good candi-
dates for a number of diverse therapeutic combinations.
WEE1 inhibitors from bench to bedside
Several clinical studies are currently evaluating the effi-
cacy of adavosertib on different aggressive and advanced
tumors (Table 4).
The results of phase I trials showed that adavosertib is
well tolerated both in single agent and in combination.
Depending on the study, the maximum tolerated dose
(MTD) was established between 150 and 225 mg orally
twice per day for 2.5 days per 2 weeks [156–158]. The
most common adverse events reported in the abovemen-
tioned studies were fatigue, nausea, vomiting, diarrhea,
and hematologic toxicity. Moreover, correlative studies
performed on tumor biopsies confirmed in vivo the mech-
anism of action of adavosertib. Indeed, immunohisto-
chemistry analyses showed a reduction of phospho-CDK1
(Tyr15) and an increase of DNA damages (phospho-
γH2AX) in cancer cells [156,157].
The phase II studies confirmed that adavosertib sensi-
tizes cancer patients to different chemotherapy agents.
Interestingly, adavosertib showed efficacy when com-
bined with carboplatin in TP53-mutated ovarian cancer
patients, refractory or resistant to first-line platinum-
based chemotherapy [159]. Similar results were reported
in platinum-resistant primary ovarian cancer patients
after treatment with the combination of adavosertib and
a single chemotherapeutic agent (carboplatin, paclitaxel,
gemcitabine, or pegylated liposomal doxorubicin) [160].
Primary resistance and predictive markers of
response to WEE1/PKMYT1 based therapies
Several DDR inhibitors have proved their efficacy against
different cancer types in the preclinical and clinical set-
tings [161–165]. Among them, WEE1 inhibitor seems to
be the most effective ones, also favored by a relative low
off-target toxicity. However, despite the number of stud-
ies and the promising results, few predictive markers of
response have been identified. Recently, cyclin E level
has been linked to the efficacy of adavosertib in breast
cancer models [89], with cyclin E-high cells, that gener-
ally show elevated chromosome instability, being more
sensitive compared with cyclin E-low ones. Despite the
reported low levels of WEE1 expression in breast cancer,
chromosome instability, that has also prognostic poten-
tial mainly in grade 2 tumors [89], may explain the
effectiveness of WEE1 inhibitors, as supported by the
predictive role of cyclin E. Our group and others showed
that high PKMYT1 expression associates with reduced
sensitivity to adavosertib, indicating a potential compen-
satory effect [35,166]. Moreover, high-throughput
proteomic profiling demonstrated that small cell lung
cancer and ovarian cancer models with primary resist-
ance to adavosertib express high levels of AKT/mTOR
pathway molecules and phosphorylated S6 ribosomal
protein [137,138]. In acute leukemia models, the sensi-
tivity to adavosertib has been recently linked to HDAC
and MYC regulation. Indeed, by generating adavosertib-
resistant models, the researchers found that resistant
acute leukemia cell lines are dependent on increased
HDAC activity for their survival, partly due to increased
KDM5A function. In addition, gene expression analyses
demonstrated a HDAC-dependent expression of MYC in
the adavosertib-resistant cell lines [167]. These observa-
tions support the success of preclinical studies combining
WEE1 and HDAC [41,130,131] or bromodomain inhibi-
tors [150].
Conclusion
Thanks to a constantly growing amount of preclinical and
clinical data, our knowledge on cancer biology is increasing
and, consequently, the list of cancer hallmarks has been
progressively expanding. Recent findings demonstrated that
cancer cells are characterized by functional and molecular
alterations in crucial genes involved in the DDR pathway,
which is fundamental for cell cycle regulation, DNA dam-
ages recognition, and repair. Functional alterations of DDR-
gene have a deep impact on tumor progression and on the
clinical outcome of cancer patients. Indeed, the efficacy of
Ghelli Luserna di Rorà et al. Journal of Hematology & Oncology (2020) 13:126 Page 10 of 17
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Table 4 Clinical trials evaluating WEE1/PKMYT1 inhibitor in monotherapy or in combination for cancer therapy
Study ID Study title Tumor Interventions Status Phase
NCT02610075 Phase Ib Study to Determine MTD of AZD1775 Monotherapy in
Patients With Locally Advanced or Metastatic Solid Tumours.
S AZD1775 C 1
NCT03668340 AZD1775 in Women With Recurrent or Persistent Uterine
Serous Carcinoma
S AZD1775 R 2
NCT02482311 Safety, Tolerance, PK, and Anti-tumour Activity of AZD1775
Monotherapy in Patients With Advanced Solid Tumours
S AZD 1775 C 1
NCT02207010 A Phase 0 Study of AZD1775 in Recurrent GBM Patients S AZD1775 NA 1
NCT03315091 Phase I Study to Assess the Effect of Food on AZD1775
Pharmacokinetics in Patients With Advanced Solid Tumours
S AZD1775 C 1
NCT01748825 AZD1775 for Advanced Solid Tumors S/H AZD1775 ANR 1
NCT02511795 AZD1775 Combined With Olaparib in Patients With Refractory
Solid Tumors
S AZD1775 + Olaparib C 1
NCT03313557 AZD1775 Continued Access Study to Assess Safety and
Tolerability for Patients Enrolled in AZD1775 Clinical
Pharmacology Studies
S AZD1775 C 1
NCT02593019 Phase II, Single-arm Study of AZD1775 Monotherapy in
Relapsed Small Cell Lung Cancer Patients
S AZD1775 NA 2
NCT02688907 Phase II, Single-arm Study of AZD1775 Monotherapy in Relapsed
Small Cell Lung Cancer Patients With MYC Family Amplification
or CDKN2A Mutation Combined With TP53 Mutation
S AZD1775 T 2
NCT02087176 A Placebo Controlled Study Comparing AZD1775 + Docetaxel
Versus Placebo + Docetaxel to Treat Lung Cancer
S AZD1775 + Docetaxel T 2
NCT03012477 CISPLATIN + AZD-1775 In Breast Cancer S AZD1775 + Cisplatin ANR 2
NCT02341456 Phase Ib Study AZD1775 in Combination With Carboplatin and
Paclitaxel in Adult Asian Patients With Solid Tumours
S AZD1775 + Carboplatin
or Paclitaxel
C1
NCT02791919 Wee1 Kinase Inhibitor AZD1775 and Combination Chemotherapy
in Treating Children, Adolescents and Young Adults With
Relapsed or Refractory Acute Myeloid Leukemia
H AZD1775 + Cytarabine
or Filgrastim
or Fludarabine Phosphate
W1
NCT02513563 AZD1775 Plus Carboplatin-Paclitaxel in Squamous Cell Lung Cancer S AZD1775 + Carboplatin
or Paclitaxel
R2
NCT03718143 AZD1775 in Advanced Acute Myeloid Leukemia, Myelodysplastic
Syndrome and Myelofibrosis
H AZD1775 + Cytarabine T 2
NCT02585973 Dose-escalating AZD1775 + Concurrent Radiation + Cisplatin for
Intermediate/High Risk HNSCC
S AZD1775 + Cisplatin +
Radiation
R1
NCT02087241 Ph II Trial of Carboplatin and Pemetrexed With or Without
AZD1775 for Untreated Lung Cancer
S AZD1775 + pemetrexed
or carboplatin
T2
NCT02381548 Phase I Trial of AZD1775 and Belinostat in Treating Patients With
Relapsed or Refractory Myeloid Malignancies or Untreated Acute
Myeloid Leukemia
H AZD1775 + Belinostat T 1
NCT03333824 Effects of AZD1775 on the PK Substrates for CYP3A, CYP2C19,
CYP1A2 and on QT Interval in Patients With Advanced Cancer
S AZD1775 C 1
NCT02906059 Study of Irinotecan and AZD1775, a Selective Wee 1 Inhibitor, in
RAS or BRAF Mutated, Second-line Metastatic Colorectal Cancer
S AZD1775 + Irinotecan R 1
NCT02037230 Dose Escalation Trial of AZD1775 and Gemcitabine (+Radiation)
for Unresectable Adenocarcinoma of the Pancreas
S AZD1775 + Gemcitabine+
Radiation Therapy
C 1,2
NCT02617277 Safety, Tolerability and Pharmacokinetics of AZD1775
(Adavosertib) Plus MEDI4736 (Durvalumab) in Patients With
Advanced Solid Tumours
S AZD1775 + Durvalumab ANR 1
NCT02666950 WEE1 Inhibitor AZD1775 With or Without Cytarabine in Treating
Patients With Advanced Acute Myeloid Leukemia or
Myelodysplastic Syndrome
H AZD1775 + Cytarabine C 2
NCT01047007 A Dose Escalation Study of MK1775 in Combination With 5-FU or
5-FU/CDDP in Patients With Advanced Solid Tumor (1775-005)
S AZD1775 + 5-FU or
5-FU/CDDP
T1
NCT01164995 Study With Wee-1 Inhibitor MK-1775 and Carboplatin to Treat S AZD1775 + carboplatin NA 2
Ghelli Luserna di Rorà et al. Journal of Hematology & Oncology (2020) 13:126 Page 11 of 17
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Table 4 Clinical trials evaluating WEE1/PKMYT1 inhibitor in monotherapy or in combination for cancer therapy (Continued)
Study ID Study title Tumor Interventions Status Phase
p53 Mutated Refractory and Resistant Ovarian Cancer
NCT02448329 Study of AZD1775 in Combination With Paclitaxel, in Advanced
Gastric Adenocarcinoma Patients Harboring TP53 Mutation as a
Second-line Chemotherapy
S AZD1775 + paclitaxel R 2
NCT02508246 WEE1 Inhibitor MK-1775, Docetaxel, and Cisplatin Before Surgery
in Treating Patients With Borderline Resectable Stage III-IVB
Squamous Cell Carcinoma of the Head and Neck
S AZD1775 + Cisplatin +
Docetaxel
C1
NCT03253679 AZD1775 in Treating Patients With Advanced Refractory Solid
Tumors With CCNE1 Amplification
S AZD1775 R 2
NCT01076400 A Study of MK-1775 in Combination With Topotecan/Cisplatin in
Participants With Cervical Cancer (MK-1775-008)
S AZD1775 + Topotecan or
Cisplatin
T 1,2
NCT02196168 Cisplatin With or Without WEE1 Inhibitor MK-1775 in Treating
Patients With Recurrent or Metastatic Head and Neck Cancer
S AZD1775 +Cisplatin T 2
NCT02101775 Gemcitabine Hydrochloride With or Without WEE1 Inhibitor MK-
1775 in Treating Patients With Recurrent Ovarian, Primary
Peritoneal, or Fallopian Tube Cancer
S AZD1775 + Gemcitabine ANR 2
NCT03028766 WEE1 Inhibitor With Cisplatin and Radiotherapy: A Trial in Head
and Neck Cancer
S AZD1775 + Cisplatin +
Radio therapy
ANR 1
NCT01357161 A Study of MK-1775 in Combination With Paclitaxel and
Carboplatin Versus Paclitaxel and Carboplatin Alone for
Participants With Platinum-Sensitive Ovarian Tumors With the
P53 Gene Mutation (MK-1775-004)
S AZD1775 + paclitaxel +
carboplation
C2
NCT03284385 Testing AZD1775 in Advanced Solid Tumors That Have a
Mutation Called SETD2
S AZD1775 R 2
NCT00648648 A Dose Escalation Study of MK-1775 in Combination With Either
Gemcitabine, Cisplatin, or Carboplatin in Adults With Advanced
Solid Tumors (MK-1775-001)
S AZD1775 + Gemcitabine
or Cisplatin or Carboplatin
C1
NCT02194829 Paclitaxel Albumin-Stabilized Nanoparticle Formulation and
Gemcitabine Hydrochloride With or Without WEE1 Inhibitor MK-
1775 in Treating Patients With Previously Untreated Pancreatic
Cancer That Is Metastatic or Cannot Be Removed by Surgery
S AZD-1775 + Gemcitabine
+ paclitaxel
ANR 1,2
NCT02576444 Olaparib Combinations S AZD1775 + olaparib ANR 2
NCT04197713 Testing the Sequential Combination of the Anti-cancer Drugs
Olaparib Followed by Adavosertib (AZD1775) in Patients With
Advanced Solid Tumors With Selected Mutations and PARP
Resistance, STAR Study
S AZD1775 + olaparib ANR 1
NCT01922076 Adavosertib and Local Radiation Therapy in Treating Children
With Newly Diagnosed Diffuse Intrinsic Pontine Gliomas
S AZD1775 + Radiation
Therapy
ANR 1
NCT03579316 Adavosertib With or Without Olaparib in Treating Patients With
Recurrent Ovarian, Primary Peritoneal, or Fallopian Tube Cancer
S AZD1775 + olaparib R 2
NCT02095132 Adavosertib and Irinotecan Hydrochloride in Treating Younger
Patients With Relapsed or Refractory Solid Tumors
S AZD1775 + Irinotecan or
Irinotecan Hydrochloride
R 1,2
NCT03345784 Adavosertib, External Beam Radiation Therapy, and Cisplatin in
Treating Patients With Cervical, Vaginal, or Uterine Cancer
S AZD1775 +Cisplatin +
Radiation (External Beam
Radiation Therapy)
R1
NCT01849146 Adavosertib, Radiation Therapy, and Temozolomide in Treating
Patients With Newly Diagnosed or Recurrent Glioblastoma
S AZD1775 + Radiation
Therapy + Temozolomide
R1
NCT02937818 A Phase II, Study to Determine the Preliminary Efficacy of Novel
Combinations of Treatment in Patients With Platinum Refractory
Extensive-Stage Small-Cell Lung Cancer
S AZD1775 + carboplatin ANR 2
NCT02546661 Open-Label, Randomised, Multi-Drug, Biomarker-Directed, Phase
1b Study in Pts w/ Muscle Invasive Bladder Cancer
S AZD1775 + Durvalumab ANR 1
NCT02659241 Adavosertib Before Surgery in Treating Patients With Advanced
High Grade Ovarian, Fallopian Tube, or Primary Peritoneal Cancer
S AZD1775 R 1
NCT02272790 Adavosertib Plus Chemotherapy in Platinum-Resistant Epithelial
Ovarian, Fallopian Tube, or Primary Peritoneal Cancer
S AZD1775 + Paclitaxel or
Carboplatin or
ANR 2
Ghelli Luserna di Rorà et al. Journal of Hematology & Oncology (2020) 13:126 Page 12 of 17
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standard of care chemo/radiotherapy regimens depends on
the generation of DNA damages in proliferating malignant
cells. In this scenario, the overexpression or uncontrolled
activation of DDR pathways has been showed to protect
cancer cells from the therapeutic effect of DNA damaging
agents. Moreover, a large number of preclinical studies
highlighted that cancer cells depend on the functionality of
DDR pathways in order to survive, to tolerate the replica-
tive stress induced by the high proliferative rate and to
sustain the intrinsic genetic instability. For these reasons,
selective inhibitors have been developed in order to exploit
cancer cells’dependency on DDR-gene functionality. Pre-
clinical data has proven the efficacy of DDR inhibition in
different kinds of hematological and solid tumors, both as
monotherapy and in combination with a wide number of
DNA damaging agents. Among DDR inhibitors, the most
effectiveoncearethosetargetingPARP1andWEE1family
kinases. The effectiveness of PARP1 inhibitors is however
dependent on homologous recombination (HR) repair
deficiency while WEE1 family kinases inhibitors seems to
have a widespread efficacy independently from a specific
the genetic background. Indeed, cancer cells seem to be
strictly dependent on the functionality of WEE1/PKMYT1
kinases to survive, especially those with alterations targeting
the G1 checkpoint. WEE1/PKMYT1 kinases are involved
in different biological processes and they seem to play di-
verse roles in nonmalignant and in cancer cells. Indeed,
they control cell cycle regulation and genetic stability in
nonmalignant cells and for these reasons act as tumor
suppressor genes. Conversely, their ability of promote DNA
damages repair and cell cycle control makes them act as
pseudo-oncogenes in cancer cells. Several molecular studies
showed that malignant cells have high expression level of
WEE1 and PKMYT1, which has become a good prognostic
biomarker for chemo/radiotherapy regimens. However, we
currently lack information regarding predictive markers of
response to WEE1/PKMYT1 inhibitors. Large preclinical
and clinical studies should be conducted in order to identify
specific molecular backgrounds in which the use of WEE1/
PKYMT1 inhibitors may be recommended. The identifica-
tion of molecular vulnerabilities in cancer patients will be
fundamental to design novel therapeutic regimens using
WEE1/PKMYT1 inhibitors in a chemo/radiotherapy-free,
synthetic lethality-based approach.
Abbreviations
ALL: Acute lymphoblastic leukemia; AML: Acute myeloid leukemia; BC: Breast
cancer; C: Chemotherapy; CC: Colorectal cancer; CN: Copy number;
CNA: Copy number alteration; CLL: Chronic lymphocyte leukemia;
CML: Chronic myeloid leukemia; DDR: DNA damage response; DLBCL: Diffuse
large B cell lymphoma; ES: Esophageal cancer; GC: Gastric cancer;
GL: Gliomas; GLB: Glioblastoma; HC: Hepatocellular carcinoma;
HR: Homologous recombination; HNSCC: Head and neck squamous cell
carcinoma; LC: Lung cancer; M: Monotherapy; MaM: Malignant melanoma;
MCL: Mantle cell lymphoma; MM: Multiple myeloma; MPF: Mitotic promoting
factor; N: Neuroblastoma; NSCLC: Non-small-cell lung cancer; OC: Ovarian
cancer; PC: Pancreatic cancer; R: Radiotherapy; S: Sarcomas; SAC: Spindle
assembly checkpoint; TNBC: Triple-negative breast cancer
Acknowledgements
Not applicable.
Authors’contributions
AGLDR, CC, and GS drafted the first version of the manuscript and created
the figures. GM critically revised the manuscript for important intellectual
content. All authors read and approved the final manuscript.
Funding
This work was supported by ERA-Per-Med (reference number: ERAPERME
D2018-275).
Availability of data and materials
Not applicable.
Ethics approval and consent to participate
Not applicable.
Consent for publication
All authors read and approved the final manuscript.
Competing interests
GM has competing interests with Novartis, BMS, Roche, Pfizer, ARIAD, and
MSD. The other authors declare that they have no competing interests.
Table 4 Clinical trials evaluating WEE1/PKMYT1 inhibitor in monotherapy or in combination for cancer therapy (Continued)
Study ID Study title Tumor Interventions Status Phase
Gemcitabine or pegylated
liposomal doxorubicin
NCT02813135 European Proof-of-Concept Therapeutic Stratification Trial of
Molecular Anomalies in Relapsed or Refractory Tumors
S/H AZD1775 + carboplatin R 1,2
NCT03330847 To Assess Safety and Efficacy of Agents Targeting DNA Damage
Repair With Olaparib Versus Olaparib Monotherapy.
S AZD1775 + olaparib R 2
NCT01827384 MPACT Study to Compare Effects of Targeted Drugs on Tumor
Gene Variations
S AZD1775 + carboplatin R 2
NCT02465060 Targeted Therapy Directed by Genetic Testing in Treating
Patients With Advanced Refractory Solid Tumors, Lymphomas,
or Multiple Myeloma (The MATCH Screening Trial)
S/H AZD1775 R 2
Ssolid tumor, Hhematological tumor, Ccompleted, Rrecruiting, Wwithdraw, ANR active not recruiting, Tterminated, NA status unknown (last update 04/22/2020)
Ghelli Luserna di Rorà et al. Journal of Hematology & Oncology (2020) 13:126 Page 13 of 17
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Received: 18 June 2020 Accepted: 2 September 2020
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