Background

The WEE1 kinase family consists of three serine/threonine 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 regulators 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 permitting 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. Therefore, 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 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 phosphatase. 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 regulation of CDK1 has been recently confirmed by in vivo studies. Indeed, the replacement of the CDK1 inhibitory phosphorylation sites with non-phosphorylatable amino acids (CDK1T14A/Y15F) was embryonic lethal in mice [12]. Once activated, the MPF complex can phosphorylate WEE1 and PKMYT1 to promote their inactivation via different cascades [5, 13, 14]. WEE1 is phosphorylated (Ser123) by CDK1 at the onset of mitosis, thereby generating 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 regulation 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 triggered through the activation of the pre-replication complex [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 CDK1T14A/Y15F induced replication stress and S phase arrest in mouse embryonic fibroblasts (MEFs), with substantial increase of γH2AX levels, chromosomal fragmentation, and DDR activation, as a consequence of intra-S phase DNA damage [12]. Moreover, 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 (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 slippage [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 spindle 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/CCdc20 activation and chromosome segregation [24,25,26]. Moreover, WEE1 directly interacts with APC/C components, including 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].

Fig. 1
figure 1

WEE1 and PKMYT1 biological functions. a Schematic 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. b Schematic 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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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 heterozygous 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 development [21]. Biological processes such as DNA replication and homologous recombination involve the formation of branched DNA structures that physically link chromosomes. Such DNA structures needs to be disengaged prior to entry into mitosis, in order to ensure proper chromosome segregation. Eukaryotic cells evolved different 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 generation 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 endonucleases 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 regulation 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 molecular and functional alterations of WEE1 and PKMYT1 in hematological and solid tumors.

Fig. 2
figure 2

WEE1 family proteins role as tumor suppressors or pseudo-oncogenes in non-malignant and cancer cells

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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 heterogeneous 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 adenocarcinoma, Fig. 3a, b). In particular, WEE1 mutations have been reported in 7.6% of UCEC cases. Moreover, PKMYT1 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), prostate adenocarcinoma (PRAD), and sarcoma (SARC), with a frequency lower than 0.5%. Both genes are mainly targeted 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 sequence (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 adenocarcinoma (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 chromosomal 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.

Fig. 3
figure 3

WEE1 and PKMYT1 mutations and copy number alterations (CNAs) in cancer. a Frequency of patients with WEE1 or b PKMYT1 gene mutations across cancers from TCGA cohorts. c Distribution of mutations according to the WEE1 and d PKMYT1 amino acid (aa) sequence and protein domains (WEE1 transcript ENST00000450114, 646 aa; PKMYT1 transcript ENST00000262300, 499 aa). e Frequency of patients with copy number gain or loss in WEE1 or f PKMYT1 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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WEE1 and PKMYT1 functional role in hematological and solid tumors

Few studies have analyzed WEE1 and PKMYT1 expression in hematological malignancies. Our group showed that WEE1 kinase is highly expressed in acute lymphoblastic 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) [36,37,38,39]. 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 combination 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, PKMYT1 proved to be essential for MM cell line viability, 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), neuroblastoma, and gastric cancers [31, 32, 45,46,47]. 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 (radiotherapy or chemotherapy) [48,49,50,51]. 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 hepatocellular carcinoma and colorectal cancers, PKMYT1 regulates epithelial-mesenchymal transition (EMT), a process relevant to tumor progression, invasion, metastasis, and drug resistance, through the activation of the beta-catenin/TCF 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 pathway [53] (Table 1). 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.

Table 1 WEE1 and PKMYT1 molecular alterations in hematological and solid tumors according to literature
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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 PKMYT1 have been investigated as therapeutic targets for hematological and solid tumors. Several pharmacological inhibitors have been designed and subsequently validated 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 (chemotherapy/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 progressive 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 sensitivity 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 discrepancies may be linked to the intrinsic chromosomal instability of the analyzed tumors and to additional alterations deregulating the G1 checkpoint in TP53 wild-type cases that may enhance the sensitivity to WEE1 targeting.

Fig. 4
figure 4

Mechanism of action of WEE1/PKMYT1 inhibitors for the treatment of cancer cells. a Schematic 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. b Schematic 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

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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, methotrexate, cisplatin, clofarabine, etoposide, 5-fluorouracil, and radiotherapy) and different WEE1/PKMYT1 inhibitors in preclinical models [48, 56, 65,66,67,68,69]. The mechanism of action of the combination is based on the inhibition of the DDR pathway following induction of DNA damage induced 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 activation 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 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.

Table 2 Effects of standard of care chemo/radiotherapy agents on cell cycle checkpoints activation
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Preclinical studies of WEE1 and PKMYT1 inhibitors

Several targeted compounds showed an inhibitory activity on WEE1 and PKMYT1 kinases and their efficacy was proven in a number of tumor types. Table 3 shows the main preclinical studies that used WEE1/PKMYT1 inhibitors in single agent or in combination with chemo/radiotherapy agents in different tumor types.

Table 3 Preclinical studies evaluating the effect of WEE1 inhibitors in monotherapy or in combination with chemotherapy/radiotherapy in cancer
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PD0166285 is the first reported drug, with an inhibitory 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 associated with a progressive accumulation of DNA damages and by the induction of apoptosis [35, 99, 119,120,121,122]. This last event is cell cycle phase-dependent and can occur (i) as a consequence of S phase checkpoint override, when cancer cells start DNA replication even in the presence of DNA damages (replicative catastrophe); (ii) following G2/M phase checkpoint override, that results in forced entry into mitosis, even in the presence 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, 127,128,129]. 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 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], CHK1 [103, 140,141,142], ATM [132,133,134,135], 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 [136,137,138,139], and proteasome inhibitors [36].

We have recently reported synergistic effects of adavosertib 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 bosutinib isomer. In the study, we speculated that the strong cytotoxic effect of the combination was due to the concomitant inhibition of WEE1 and PKMYT1 kinases [35]. Indeed, no selective inhibitor has been currently developed to target its functionality. However, several known tyrosine kinase inhibitors have an inhibitory off-target effect on PKMYT1. Among them, compounds commonly used for the treatment of BCR-ABL1-positive CML and ALL, as dasatinib and bosutinib (and a structural isomer of bosutinib [154, 155]) were shown to inhibit PKMYT1 activity.

Overall, the data suggest that WEE1/PKMYT1 inhibition is a suitable pharmacological target for combination strategies in cancer. The broad spectrum of activities exerted by the two kinases, and especially by WEE1, across the cell cycle, makes them good candidates for a number of diverse therapeutic combinations.

WEE1 inhibitors from bench to bedside

Several clinical studies are currently evaluating the efficacy of adavosertib on different aggressive and advanced tumors (Table 4).

Table 4 Clinical trials evaluating WEE1/PKMYT1 inhibitor in monotherapy or in combination for cancer therapy
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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 [156,157,158]. The most common adverse events reported in the abovementioned studies were fatigue, nausea, vomiting, diarrhea, and hematologic toxicity. Moreover, correlative studies performed on tumor biopsies confirmed in vivo the mechanism of action of adavosertib. Indeed, immunohistochemistry 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 sensitizes cancer patients to different chemotherapy agents. Interestingly, adavosertib showed efficacy when combined 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 settings [161,162,163,164,165]. 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 studies 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 generally 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 potential 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 compensatory effect [35, 166]. Moreover, high-throughput proteomic profiling demonstrated that small cell lung cancer and ovarian cancer models with primary resistance to adavosertib express high levels of AKT/mTOR pathway molecules and phosphorylated S6 ribosomal protein [137, 138]. In acute leukemia models, the sensitivity 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 observations support the success of preclinical studies combining WEE1 and HDAC [41, 130, 131] or bromodomain inhibitors [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 damages 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 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 replicative 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 effective once are those targeting PARP1 and WEE1 family 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 diverse 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 identification 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.