Introduction

Oncology Reports

1021-335X 1791-2431

D.A. Spandidos

10.3892/or.2023.8562

OR-49-6-08562

Articles

Combination of PARP and WEE1 inhibitors in vitro: Potential for use in the treatment of SHH medulloblastoma

Lukoseviciute

Monika

Theodosopoulou

Aikaterini

Holzhauser

Stefan

Dalianis

Tina

Kostopoulou

Ourania N.

Department of Oncology-Pathology, Karolinska Institutet, 171 64 Stockholm, Sweden

Correspondence to: Dr Ourania N. Kostopoulou, Department of Oncology-Pathology, Karolinska Institutet, Bioclinicum J6:20, 171 64 Stockholm, Sweden, E-mail: minami@kobe-u.ac.jp ourania.kostopoulou@ki.se

06 2023

05 05 2023

49 6

125

19012023 28032023

2023

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Medulloblastoma (MB), grouped as either WNT-activated, Sonic hedgehog (SHH)-activated, or non-WNT/non-SHH group 3, accounts for almost 20% of all childhood brain cancers. In spite of current intensive treatments, not all patients are cured and survivors suffer from severe side-effects. The present study therefore examined the effects of the poly-ADP-ribose polymerase (PARP) and WEE1-like protein kinase (WEE1) inhibitors, BMN673 and MK-1775, respectively, alone or in combination on four MB cell lines. More specifically, the MB cell lines, DAOY, UW228-3, MED8A and D425, were tested for their sensitivity to BMN673 and MK-1775 alone or in combination, using cell viability, cell confluency and cytotoxicity assays. The effects on the cell cycle phases were also examined using FACS analysis. Monotherapy with BMN673 and MK-1775 exerted dose-dependent inhibitory effects on the viability of almost all MB cell lines. Notably, when BMN673 and MK-1775 were used in combination, synergistic effects were noted in the SHH group cell lines (DAOY and UW228-3), but not in the already WEE1-sensitive group 3 (MED8A and D425) lines. Moreover, the combination treatment decreased the percentage of cells in the G1 phase and induced the novel distribution of both DAOY and UW228-3 cells in the S and G2/M phases, with the UW228-3 cells exhibiting a greater delay. To conclude, MK-1775 was efficient in all and BMN673 in most cell lines, and their combined use exerted synergistic effects on the SHH, but not the group 3 cell lines. These data suggest that MK-1775 alone may be of interest for all MB cell lines, and that the combination of PARP/WEE1 inhibitors may provide possible therapeutic opportunities for the therapy of SHH MBs. Their use warrants further investigations in the future.

medulloblastoma WEE1 inhibitors poly(ADP-ribose) polymerase inhibitors DAOY UW228-3

Swedish Childhood Cancer Foundation

TJ2022-0067

Swedish Cancer Foundation

20 0704

Stockholm Cancer Society

201092

Stockholm City Council

20180037

AnnaBrita o Bo Casters Minne Foundation (Lindhés Advokatbyrå)

LA2022-0070

Svenska Läkaresällskapet

SLS-934161

Åke Wiberg Foundation

M21-0012

Karolinska Institutet Sweden

2022-01587

Tornspiran Foundation

839

The present study was supported by the Swedish Childhood Cancer Foundation (grant no. TJ2022-0067), the Swedish Cancer Foundation (grant no. 20 0704), the Stockholm Cancer Society (grant no. 201092), the Stockholm City Council (grant no. 20180037), AnnaBrita o Bo Casters Minne Foundation (Lindhés Advokatbyrå) (grant no. LA2022-0070), Svenska Läkaresällskapet (grant no. SLS-934161), Åke Wiberg Foundation (grant no. M21-0012) Karolinska Institutet Sweden (grant no. 2022-01587), and Tornspiran Foundation (grant no. 839).

Introduction

Medulloblastoma (MB) is a highly malignant and invasive childhood brain tumor most predominantly located in the cerebellum (1–4). Currently, the majority of patients with MB undergo surgical resection and receive multi-agent chemotherapy, while children ˃3 years of age are also treated with craniospinal radiation therapy (5,6). Although current treatment strategies have increased the average survival of rate of patients with MB up to 70%, the major disadvantages of such therapies include the development of drug resistance, metastasis, disease recurrence which is universally fatal, and long-term toxicities (7–9). There is therefore a need for the development of novel personalized targeted therapies focusing on molecular alterations and individual tumor molecular profiles.

According to the renewed 2021 World Health Organization (WHO) Classification of Tumor of the Central Nervous System, MB is classified into four genetically-defined subgroups, including WNT-activated (best prognosis), Sonic hedgehog (SHH)-activated TP53 wild-type, SHH-activated TP53-mutant, and non-WNT/non-SHH (group 3, worst prognosis; and group 4, intermediate outcomes) (10–17).

The understanding of specific tumorigenic mutations, molecular drivers and deregulated signaling pathways in molecular subgroups accelerates the discovery of potential therapeutic targets. Potential targets are poly(ADP-ribose) (PAR) polymerase (PARP)1 and PARP2, which are known as the key proteins coordinating the DNA damage response, specifically the damage detection and repair of both single- and double-strand breaks (18–21). In particular, PARP1 and PARP2 (PARP1/2) are involved in the synthesis of PAR chains, which in turn recruit DNA repair proteins. PARP inhibitors have therefore been actively used in the treatment of cancers harboring defects in homologous recombination, such as in tumors with BRCA1 and BRCA2 mutations, thereby leading to synthetic lethality (22,23). Among the PARP inhibitors, BMN673 (talazoparib) is the most potent selective PARP1/2 inhibitor (24,25). BMN673 is currently approved by the US Food and Drug Administration (FDA) for the treatment of BRCA1 or BRCA2 mutated, negative HER2 locally advanced or metastatic breast cancer (26). In other clinical studies, an ongoing phase II trial is testing the effects of BMN673 in patients with recurrent high-grade glioma (NCT04740190). Although BMN673 has not extensively been tested in MB, previous studies have demonstrated that other PARP inhibitors, such as olaparib, rucaparib and veliparib are potential chemo- and radiosensitizing agents in MB cells and xenograft models (27–29).

Another promising molecular target is the WEE1-like protein kinase (WEE1), which is involved in the regulation of the S phase and G2/M checkpoint of the cell cycle (30–32). In response to DNA damage, WEE1 mediates the inhibition of the phosphorylation of cyclin-dependent kinase (CDK) 1 and CDK2, resulting in cell cycle arrest and possible DNA repair (33,34). Multiple studies have reported increased expression levels of WEE1 in various types of cancer, including pediatric high-grade gliomas, glioblastoma, ovarian cancer, melanoma, breast cancer and MB (33,35–38). The inhibition of WEE1 can impair the G2/M checkpoint, allowing cancer cells with DNA damage to divide, eventually leading to mitotic catastrophe (39). Currently, MK-1775 is the first selective small-molecule inhibitor of WEE1, which has exhibited promising antitumor efficacy, when combined with chemotherapeutics agents, against pancreatic, breast, colon and ovarian cancer (40–43). In addition, it has been reported that MK-1775 is highly effective in tumor cells harboring p53 mutations (44,45). Specifically, in MB, previous research has demonstrated that MK-1775 single treatment can inhibit MB tumor growth in vivo (46).

The crucial roles of PARP and WEE1 in the DNA damage response render them potential therapeutic targets against MB tumors. The present study focused on targeting proteins that are critical for DNA damage repair, potentially making this treatment strategy applicable to different molecular subgroups. More specifically, the present study examined the effects of the FDA-approved PARP inhibitor, BMN673, and the not yet FDA-approved WEE1 inhibitor, MK-1775, as single agents and in combination on four MB cell lines DAOY, UW228-3, MED8A and D425.

Materials and methods <sec> <title>Tumor cell lines and cell seeding

The MB cell lines DAOY, UW228-3 (both group SHH) and D425 and MED8A (both group 3), were obtained from Professor Per Kogner, Karolinska Institutet and cultured in minimum essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM):nutrient mixture F-12 (DMEM/F-12) and DMEM with GlutaMAX (both Group 3 cell lines), respectively, with the addition of 10% fetal bovine serum (FBS) (all from Gibco; Thermo Fisher Scientific, Inc.), together with 1% L-glutamine, 100 U/ml of penicillin as well as 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.). The DAOY, UW228-3 and D425 cells are p53 mutated and D425 and MED8A have a MYC amplification (47) (https://www.cellosaurus.org/CVCL_1275; http://www.cellosaurus.org/CVCL_M137). For the viability and proliferation/cytotoxicity assays, 2.5×10³ cells/well were plated for the DAOY cells, 5×10³ cells/well were plated for the UW228-3 cells, and 10⁴ cells/well were plated for the D425 and MED8A cells (group 3 were grown in suspension) in 90 and 200 µl medium in 96-well plates, respectively. For western blot (WB) and FACS analyses, 5×10⁵ cells/Ti25 flask in 5 ml medium were plated for both the DAOY and UW228-3 MB cell lines.

Inhibitors

The PARP inhibitor, BMN673 (talazoparib), the WEE1 inhibitor, MK-1775 (AZD-1775, adavosertib), the phosphoinositide 3-kinase (PI3K) inhibitor, BYL719 (alpelisib), and the CDK4/6 inhibitor, PD-0332991 (palbociclib), in DMSO stock solutions (Selleck Chemicals GmbH), were used in various dilutions in PBS [further details have been previously described (47,48)].

WST-1 viability assay

Following the addition of (0.1, 0.5, 1 and 10 MK-1775 and BMN673 and their combinations), for 24, 48 and 72 h, the viability, i.e., the estimation of remaining live healthy cells in response to therapy of the cell population was estimated using WST-1 viability assay (Roche Diagnostics GmbH) as previously described in more detail (47,48).

Cell confluency, cytotoxicity and apoptosis assays

The IncuCyte S3 Live Cell Analysis System was used to examine the cell confluency, as a measure of proliferation following treatment and cytotoxicity, as a measure of cell damage following treatment of the DAOY and UW228-3 cells, both grown as monolayers (47,48). More specifically, at 24 h after seeding, the medium was changed to a new medium containing the Incucyte™ Cytotox Red Reagent (Essen Bioscience), that enters the damaged plasma membrane and binds to DNA in the nuclei, the treatments were then added and the plates were incubated at 37°C in the machine for 72 h. Images were collected every 2 h to follow cell confluence/proliferation with IncuCyte S3 Live Cell Analysis System (Satorius). Cytotoxicity was quantified by counting the red nuclei. Apoptosis was also assayed in the DAOY and UW228-3 cells using the IncuCyte S3 Live Analysis System by the addition of the IncuCyte Caspase-3/7 Green Apoptosis reagent, that enters live cells, as previously described in further detail (47,48).

FACS analysis

For cell cycle analysis, the cells were collected following 48 h of treatment and fixed with 70% ethanol. A total of 5×10⁵ cells were counted and stained with FxCycleR/RNAse solution (Invitrogen™, ThermoFisher Scientific, Inc.). All samples were analyzed with the FACS NovoCyte 3000, while the analysis of the data was conducted using FlowJo_v10.8.1 software (BD Biosciences).

Statistical analysis

All the results were subjected to statistical analysis. To estimate the efficacy of the single or combination treatments compared to the negative control, a multiple t-test accompanied by the correction for multiple comparisons of the means using the Holm Sidak method were used as previously described (49). To investigate the efficacy of the drug combinations the Synergy FinderPlus computational tool (https://synergyfinderplus.org/#!/) with the highest single agent (HSA) was used. HSA values >10 indicated synergistic effects of the drugs, HSA values from −10 to 10 indicated additive effects, and HSA values <-10 indicated antagonism (50).

Results <sec> <title>Effects of single and combination drug treatment with WEE1 and PARP inhibitors on MB cell lines measured using WST-1 assays

All MB cell lines (DAOY, UW228-3, D425 and MED8A) exhibited a concentration-dependent inhibition of viability at 24–72 h following treatment (determined using WST-1 assays) with the WEE1 inhibitor, MK-1775 (0.1–10 µM), and likewise, with the exception of DAOY cells, with the PARP inhibitor, BMN673 (0.1–10 µM).

MK-1775

All MB cell lines exhibited a >50% significant decrease in viability in comparison to PBS control with the highest concentration (10 µΜ) of the WEE1 inhibitor, MK-1775, at almost all time points following treatment (for all at least P<0.005) (Fig. 1A-D). In addition, all MB cell lines, apart from the UW228-3 cells, exhibited a >50% decrease in viability compared to the PBS control following treatment with 0.5 and 1 µΜ MK-1775 at most time points (for all those at least P<0.05) (Fig. 1A-D).

BMN673

Only the highest concentration (10 µΜ) of the PARP inhibitor, BMN673, led to a >50% decrease in viability in comparison to the PBS control at 72 h following treatment in all MB cell lines, apart from the DAOY cells (for all those at least P<0.001) (Fig. 1E-H).

MK-1775 and BMN673 in combination

All MB cell lines, apart from the UW228-3 cells, exhibited a >50% decrease in viability compared to the PBS control at both 48 and 72 h following treatment with all drug combinations, while the UW228-3 cells only exhibited a significant decrease with the highest combination concentrations at 72 h following treatment (for all at least P<0.05) (Fig. 1I-L).

To summarize, all four MB cell lines exhibited concentration-dependent responses to both inhibitors with the exception of the DAOY cells to BMN673, and with MK-1775 being more efficient than BMN673 at the drug concentrations used. Moreover, upon combining the inhibitors, the UW228-3 cells were generally more resistant as compared to the other three MB cell lines.

Synergistic effects of combined drug treatment with WEE1 and PARP inhibitors on MB cell lines

Based on the cell viability data described above, the synergy between WEE1 and PARP inhibitors was examined using the Synergy FinderPlus program. To examine the synergy, the synergy scores for the four MB cell lines treated with a range of combinations of WEE1 and PARP inhibitors were calculated (Fig. 2). HSA values >10 indicated synergistic effects of the drugs, HSA values from −10 to 10 indicated additive effects, and HSA values <-10 indicated antagonism (50).

The calculations obtained revealed that synergy was most clearly found when combining MK-1775 and BMN673 in the DAOY and UW228-3 cells, while the same drug combinations exerted mainly antagonistic effects in the D425 and MED8A cells (Fig. 2). Since synergy was optimal in the UW228-3 and DAOY cells, these two cell lines were selected for use in further experiments.

Other combinations

MK-1775 was also combined with BYL719 and PD-0332991, two other regulators of the cell cycle, in order to examine the joint effects of MK-1775 with BYL719 or PD-0332991 on DAOY and UW228-3 cells. However, at the concentrations used (5 and 10 µΜ of BYL719, and 5 and 10 µΜ of PD-0332991), no major enhancements were observed as compared to the effect of MK-1775 alone, with possibly one exception of a slight enhancement using MK-1775 and PD-0332991 in combination on the UW228-3 cells (data not shown). These data were therefore not pursued further herein.

Cell confluency and cytotoxicity following treatment of the DAOY and UW228-3 cells with WEE1 and PARP inhibitors either alone or in combination

The effects of treatment with the MK-1775 and BMN673 inhibitors either alone or in combination on the DAOY and UW228-3 cells were further analyzed up to 72 h following treatment using cell confluency and cytotoxicity assays utilizing the IncuCyte S3 Live-Cell Analysis System (Figs. 3 and 4).

Cell confluency MK-1775

For the DAOY cells, both the MK-1775 concentrations (0.5 and 1 µΜ) used induced an almost complete reduction in cell confluency as compared to the PBS control, while for the UW228-3 cells, only a marginal reduction in cell confluency was noted (Fig. 3A and D).

BMN673

For the DAOY cells, both concentrations (0.5 and 1 µΜ) of BMN673 used resulted in a reduced cell confluency compared to the PBS control, while for the UW228-3 cells, no effect on cell confluency was observed (Fig. 3B and E).

MK-1775 and BMN673

For the DAOY cells, all drug combinations resulted in an almost complete reduction of cell confluency compared to the PBS control, while for the UW228-3 cells, the effects were concentration-dependent and less pronounced (Fig. 3C and F).

To conclude, MK-1775 alone considerably reduced the confluency of the DAOY cells and this was also the case, to a certain extent, for cell confluency upon treatment with BMN673; an enhanced was not detected for the DAOY cells following combination treatment. By contrast, while almost no effect on UW228-3 cell confluency was observed upon treatment with the inhibitors alone, when used in combination, the inhibitors led to a concentration-dependent inhibition of cell confluency.

Cytotoxicity MK-1775

Both the MK-1775 concentrations (0.5 and 1 µΜ) exerted marked cytotoxic effects on the DAOY cells, whereas no marked cytotoxic effects were observed on the UW228-3 cells (Fig. 4A and D).

BMN673

Both the BMN673 concentrations (0.5 and 1 µΜ) exerted some cytotoxic effects on the DAOY cells, whereas no cytotoxic effects were observed on the UW228-3 cells (Fig. 4B and E).

MK-1775 and BMN673

The combined use of MK-1775 with BMN673 did not exert any exerted enhanced cytotoxic effects on either the DAOY or UW228-3 cells as compared to using the most efficient single inhibitor, MK-1775 (Fig. 4C and F).

To investigate this further, and to determine whether part of the cytotoxic response was due to apoptosis, an apoptosis assay was also performed. However, no major effects on apoptosis were observed with any of the single or combined drug administrations used above (Fig. S1).

To conclude, although treatment with MK-1775, but also BMN673 alone exerted cytotoxic effects on the DAOY cells, their combined use did not exert any enhanced effects. In the UW228-3 cells, none of the single or combined treatments exerted pronounced cytotoxic effects.

Effects of PARP and WEE1 inhibitors alone or in combination on the cell cycle progression of MB cells

The effects on cell cycle progression were examined using a FACS NovoCyte 3000 machine and FlowJo_v10.8.1 software. More specifically, the effects of WEE1 and PARP inhibitors alone or in combination (MK-1775 and BMN673) were examined on the SHH cell lines, DAOY and UW228-3 (Figs. 5 and S2).

MK-1775

Both single MK-1775 concentrations (0.5 and 1 µΜ) induced an increase in the proportion of cells in the S and G2 phases (compared to the PBS control), with a higher percentage of cells arrested in the G2 phase using the 1 µΜ concentration in both cell lines, although with a higher amount of UW228-3 cells remaining in the G1 phase in comparison to the DAOY cells (Fig. 5).

BMN673

Both single BMN673 concentrations (0.5 and 1 µΜ) induced an increase in the proportion of cells in the S phase and to a lower extent in the G2 phase (compared to the PBS control) in both cell lines, although with a higher number of UW228-3 cells remaining in the G1 phase in comparison to the DAOY cells (Fig. 5).

MK-1775 and BMN673

All MK-1775 and BMN673 combinations, irrespective of the concentrations used, led to a decrease in the percentage of cells in the G1 phase, with a clear shift of the cells from the S to the G2 and >G2 phases in both the DAOY and UW228-3 (Fig. 5).

To conclude, the single MK-1775 and BMN673 administrations increased the proportion of cells in the S and G2 phases in both cell lines, and their combination exerted additive effects on both cell lines; however, the shifts in cell cycle progression were generally delayed in the UW228-3 cells when compared to the DAOY cells.

Discussion

In the present study, WEE1 and PARP inhibitors (MK-1775 and BMN673, respectively) were evaluated alone or in combination in four MB cell lines, namely DAOY, UW228-3 (both group SHH), and D425 and MED8A (both group 3). All MB cell lines exhibited, at the concentrations used, concentration-dependent responses, with a decrease in viability upon single MK-1775 treatment and with the exception of the DAOY cells, which to certain to a certain extent, also exhibited a response to BMN673 treatment. Furthermore, upon combining the two drugs, synergy was noted in the DAOY and UW228-3 cells (both group SHH), but not in the group 3 cell lines; thus, only the former two cell lines were examined in further detail for the effects of single and combined inhibitor treatments on cell confluency, cytotoxicity and the cell cycle.

Presently, the authors have no explanation as to why synergy with regard to viability upon combination treatments with MK-1775 and BMN673 was not obtained in group 3 MB cell lines. One could have argued that this could have been due to the fact that the two SHH cell lines, DAOY and UW228-3, have p53 mutations; however, this was also the case for the D425 cells; thus, obviously, this is an issue that warrants further investigation in future studies.

Notably, at the concentrations used, MK-1775 alone was superior to BMN673 upon single treatments as regards its effects on viability in all cell lines. A similar outcome was also observed in cell confluency and cytotoxicity, where the DAOY cells were more sensitive than the UW228-3 cells, which in turn exhibited marginal effects with regard to both cell confluency and cytotoxicity. The data obtained herein for BMN673 are thereby in line with previous data on BMN673 in other childhood cancers; a previous study demonstrated limited clinical activity and suggested that single treatment with BMN673 possibly would be more efficient in patients whose tumors had defects in homologous recombination repair (51).

When MK-1775 and BMN673 were used in combination, synergy was disclosed, particularly as mentioned above, in viability, in which a reduction was observed in both the DAOY and UW228-3 cells, while the synergistic effects on cell confluency mainly applied to the UW228-3 cells, where the single drugs had limited effects. On the other hand, a synergistic effect on cytotoxicity was not detected in any cell line, although the DAOY cells were generally more sensitive than the UW228-3 cells, corresponding to similar findings from previous research by the authors using other inhibitors (47).

The concentration-dependent effects which were observed on the MB cell lines with single MK-1775 treatments were expected, since this has been previously demonstrated with corresponding inhibitors on MB and other tumor cell lines, such as head and neck cancer (46,52–55).

Previous analysis with other PARP inhibitors, such as olaparib, rucaparib and veliparib has shown that they are potential chemo- and radiosensitizing agents in MBs, even though there are, to the best of our knowledge, no other publications available using BMN673 on MB (27–29). Furthermore, to the best of our knowledge, there are no other studies available examining the MK-1775 and BMN673 combinations in MB cell lines. Nevertheless, it is known that WEE1 inhibition is efficient in cell lines exhibiting p53 mutations, although the response is not always only related to a p53 mutation alone (56).

In addition, in the present study, as mentioned above in the two SHH MB cell lines, the effects on cell cycle progression were examined, since both MK-1775 and BMN673 induce G2/M cell cycle arrest (30–32,57). Following single MK-1775 and BMN673 treatments, there was a decrease in the proportion of cells in the G1 phase in both the DAOY and UW228-3 cells, and an increase in the proportion of cells shifting towards the S and G2/M phase, which is in accordance with recent study, particularly for MK-1775, since it exerts cytotoxic effects in both the S and G2/M phase (56).

Combined treatment with MK-1775 and BMN673 exerted synergistic effects on both cell lines and further decreased the proportion of cells in the G1 and S phases, and increased the number of cells in the G2/M phase. Of note, combined treatment in both cell lines resulted in a clear increase in the proportion of cells in the >G2 phase; this is not surprising, as previous studies on MK-1775 have suggested that WEE1 inhibition causes an impairment in cytokinesis, leading to tetraploid cells (55). This needs to be examined further in order to validate the obtained results. When comparing DAOY and UW228-3 with regard to shifting towards the G2/M phase, the responses of the UW228-3 cells were generally more delayed as compared to those of the DAOY cells. The reason for this currently remains unknown, apart from the fact that the UW228-3 cells are generally more resistant than the DAOY cells (47,48).

Notably, since it has been previously demonstrated that targeted therapy may cause problems in G1 control and can lead to cancer cells becoming dependent on the G2 control to repair DNA damage (58), targeting the G2 checkpoint could be proposed as a possible additional anticancer strategy. Based on the aforementioned hypothesis, the present study combined the WEE1 inhibitor, MK-1775, with the FDA-approved PI3K and CDK4/6 inhibitors, BYL719 and PD-0332991; however, at the concentrations used, only slight synergistic effects were observed with MK-1775 and PD-0332991 on the UW228-3 cells (data not shown); thus, this was not pursued further herein.

There were some limitations to the present study, since only a small number of inhibitors and cell lines were used. Nevertheless, of note, the obtained data demonstrate that drug-drug interactions using WEE1 and PARP inhibitors are complex and can result in either synergistic or antagonistic interactions, depending on the MB subgroup profile and the mutation profile of the different cell lines. While broader concentration ranges and modified incubated periods may shed further light on the drug interactions with respect to their antitumor efficacy, the concentrations used herein adhere to commonly used standard conditions, and therefore allow for more direct comparisons (47,55).

Further and more detailed studies are warranted in order to disclose the possible mechanisms underlying the tested drug combinations exerting synergistic or antagonistic effects and to provide a pre-clinical rationale of how to apply the corresponding combinations clinically. Nevertheless, combining the WEE1 inhibitor, MK-1775, and the PARP inhibitor, BMN673, in two SHH MB cell lines, exerted synergistic effects and allowed for the use of lower inhibitor concentrations compared to those of single treatments, and this could possibly reduce some side-effects. Moreover, targeting MB with two different mechanisms may decrease the risk of resistance.

In conclusion, the present study suggests that treatment with WEE1 alone can have effects on SHH and group 3 MB, and combining WEE1 and PARP inhibitors may be of potential interest for the treatment of the SHH MB group.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

ML, AT and SH performed the majority of the experiments, interpreted the data, performed the statistical analyses and contributed to the writing of the manuscript. ML contributed together with ONK to the graphs of the manuscript. ML and SH initiated the experiments and the interpretation of the initial experiments, and contributed to the writing of the material and methods section, all under the supervision of ONK. TD and ONK made substantial contributions to the conception and design of the study, as well as to the acquisition of data, and analysis and interpretation of data, and were also involved in the drafting of the manuscript and revising it critically for important intellectual content. TD also provided the resources for the performance of the experiments such as laboratory space and consumables. TD and ONK were responsible for obtaining financial support for conducting the research Project. All authors critically read and approved the manuscript. ML and ONK confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References 1

Northcott

Robinson

Kratz

Mabbott

Pomeroy

Clifford

Rutkowski

Ellison

Malkin

Taylor

Medulloblastoma

Nat Rev Dis Primers5112019

10.1038/s41572-019-0063-6

30765705

Mahapatra

Amsbaugh

Medulloblastoma

StatPearlsTreasure Island (FL)pp12021

Northcott

Jones

Kool

Robinson

Gilbertson

Cho

Pomeroy

Korshunov

Lichter

Taylor

Pfister

Medulloblastomics: The end of the beginning

Nat Rev Cancer128188342012

10.1038/nrc3410

23175120

Kumar

Deepa

Moinca

Suresh

Naidu

Medulloblastoma: A common pediatric tumor: Prognostic factors and predictors of outcome

Asian J Neurosurg10502015

10.4103/1793-5482.151516

25767583

Martin

Raabe

Eberhart

Cohen

Management of pediatric and adult patients with medulloblastoma

Curr Treat Options Oncol155815942014

10.1007/s11864-014-0306-4

25194927

Liu

Ding

Tan

Zhang

Medulloblastoma: Molecular understanding, treatment evolution, and new developments

Pharmacol Ther2101075162020

10.1016/j.pharmthera.2020.107516

32105673

Leary

Olson

The molecular classification of medulloblastoma: Driving the next generation clinical trials

Curr Opin Pediatr2433392012

10.1097/MOP.0b013e32834ec106

22189395

Mulhern

Palmer

Merchant

Wallace

Kocak

Brouwers

Krull

Chintagumpala

Stargatt

Ashley

Neurocognitive consequences of risk-adapted therapy for childhood medulloblastoma

J Clin Oncol23551155192005

10.1200/JCO.2005.00.703

16110011

Salloum

Chen

Yasui

Packer

Leisenring

Wells

King

Howell

Gibson

Krull

Late morbidity and mortality among medulloblastoma survivors diagnosed across three decades: A report from the childhood cancer survivor study

J Clin Oncol377317402019

10.1200/JCO.18.00969

30730781

Cotter

Hawkins

Medulloblastoma: WHO 2021 and beyond

Pediatr Dev Pathol2523332022

10.1177/10935266211018931

35168417

Louis

Perry

Wesseling

Brat

Cree

Figarella-Branger

Hawkins

Pfister

Reifenberger

The 2021 WHO classification of tumors of the central nervous system: A summary

Neuro Oncol23123112512021

10.1093/neuonc/noab106

34185076

Remke

Ramaswamy

Taylor

Medulloblastoma molecular dissection: The way toward targeted therapy

Curr Opin Oncol256746812013

10.1097/CCO.0000000000000008

24076581

Kijima

Kanemura

Molecular classification of medulloblastoma

Neurol Med Chir (Tokyo)566876972016

10.2176/nmc.ra.2016-0016

27238212

Lospinoso Severini

Ghirga

Bufalieri

Quaglio

Infante

Di Marcotullio

The SHH/GLI signaling pathway: A therapeutic target for medulloblastoma

Expert Opin Ther Targets24115911812020

10.1080/14728222.2020.1823967

32990091

Gajjar

Robinson

Medulloblastoma-translating discoveries from the bench to the bedside

Nat Rev Clin Oncol117147222014

10.1038/nrclinonc.2014.181

25348790

Northcott

Korshunov

Pfister

Taylor

The clinical implications of medulloblastoma subgroups

Nat Rev Neurol83403512012

10.1038/nrneurol.2012.78

22565209

Kool

Korshunov

Remke

Jones

Schlanstein

Northcott

Cho

Koster

Schouten-van Meeteren

van Vuurden

Molecular subgroups of medulloblastoma: An international meta-analysis of transcriptome, genetic aberrations, and clinical data of WNT, SHH, group 3, and group 4 medulloblastomas

Acta Neuropathol1234734842012

10.1007/s00401-012-0958-8

22358457

Rouleau

Patel

Hendzel

Kaufmann

Poirier

PARP inhibition: PARP1 and beyond

Nat Rev Cancer102933012010

10.1038/nrc2812

20200537

Beck

Robert

Reina-San-Martin

Schreiber

Dantzer

Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3

Exp Cell Res32918252014

10.1016/j.yexcr.2014.07.003

25017100

Mladenov

Iliakis

Induction and repair of DNA double strand breaks: The increasing spectrum of non-homologous end joining pathways

Mutat Res71161722011

10.1016/j.mrfmmm.2011.02.005

21329706

Benafif

Hall

An update on PARP inhibitors for the treatment of cancer

Onco Targets Ther85195282015

25750544

Ray Chaudhuri

Nussenzweig

The multifaceted roles of PARP1 in DNA repair and chromatin remodelling

Nat Rev Mol Cell Biol186106212017

10.1038/nrm.2017.53

28676700

Wang

Tang

Guo

The role of PARP1 in the DNA damage response and its application in tumor therapy

Front Med61561642012

10.1007/s11684-012-0197-3

22660976

Shen

Rehman

Feng

Boshuizen

Bajrami

Elliott

Wang

Lord

Post

Ashworth

BMN 673, a novel and highly potent PARP1/2 inhibitor for the treatment of human cancers with DNA repair deficiency

Clin Cancer Res19500350152013

10.1158/1078-0432.CCR-13-1391

23881923

Murai

Huang

Renaud

Zhang

Takeda

Morris

Teicher

Doroshow

Pommier

Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib

Mol Cancer Ther134334432014

10.1158/1535-7163.MCT-13-0803

24356813

Hoy

Talazoparib: First global approval

Drugs78193919462018

10.1007/s40265-018-1004-5

30506138

Buck

Dyer

PJC

Hii

Carline

Kuchibhotla

Byrne

Howlett

Whitehouse

Ebert

McDonald

Veliparib is an effective radiosensitizing agent in a preclinical model of medulloblastoma

Front Mol Biosci86333442021

10.3389/fmolb.2021.633344

33996894

van Vuurden

Hulleman

Meijer

Wedekind

Kool

Witt

Vandertop

Würdinger

Noske

Kaspers

Cloos

PARP inhibition sensitizes childhood high grade glioma, medulloblastoma and ependymoma to radiation

Oncotarget29849962011

10.18632/oncotarget.362

22184287

Daniel

Rozanska

Mulligan

Drew

Thomas

Castelbuono

Hostomsky

Plummer

Tweddle

Boddy

Central nervous system penetration and enhancement of temozolomide activity in childhood medulloblastoma models by poly(ADP-ribose) polymerase inhibitor AG-014699

Br J Cancer103158815962010

10.1038/sj.bjc.6605946

20978505

Garcia

Snedeker

Baturin

Gardner

Fosmire

Zhou

Jordan

Venkataraman

Vibhakar

Porter

A small-molecule inhibitor of WEE1, AZD1775, synergizes with olaparib by impairing homologous recombination and enhancing DNA damage and apoptosis in acute leukemia

Mol Cancer Ther16205820682017

10.1158/1535-7163.MCT-16-0660

28655785

McGowan

Russell

Cell cycle regulation of human WEE1

EMBO J14216621751995

10.1002/j.1460-2075.1995.tb07210.x

7774574

Ghelli Luserna di Rorà

Cerchione

Martinelli

Simonetti

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

J Hematol Oncol131262020

10.1186/s13045-020-00959-2

32958072

Doroshow

Kummar

WEE1 kinase as a target for cancer therapy

Cell Cycle12315931642013

10.4161/cc.26062

24013427

Jin

Morgan

Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells

J Cell Biol1349639701996

10.1083/jcb.134.4.963

8769420

Mueller

Hashizume

Yang

Kolkowitz

Olow

Phillips

Smirnov

Tom

Prados

James

Targeting Wee1 for the treatment of pediatric high-grade gliomas

Neuro Oncol163523602014

10.1093/neuonc/not220

24305702

Caretti

Hiddingh

Lagerweij

Schellen

Koken

Hulleman

van Vuurden

Vandertop

Kaspers

Noske

Wurdinger

WEE1 kinase inhibition enhances the radiation response of diffuse intrinsic pontine gliomas

Mol Cancer Ther121411502013

10.1158/1535-7163.MCT-12-0735

23270927

Magnussen

Holm

Emilsen

Rosnes

Slipicevic

Flørenes

High expression of Wee1 is associated with poor disease-free survival in malignant melanoma: Potential for targeted therapy

PLoS One7e382542012

10.1371/journal.pone.0038254

22719872

Iorns

Lord

Grigoriadis

McDonald

Fenwick

Mackay

Mein

Natrajan

Savage

Tamber

Integrated functional, gene expression and genomic analysis for the identification of cancer targets

PLoS One4e51202009

10.1371/journal.pone.0005120

19357772

Mir

De Witt Hamer

Krawczyk

Balaj

Claes

Niers

Van Tilborg

Zwinderman

Geerts

Kaspers

In silico analysis of kinase expression identifies WEE1 as a gatekeeper against mitotic catastrophe in glioblastoma

Cancer Cell182442572010

10.1016/j.ccr.2010.08.011

20832752

Hirai

Arai

Okada

Nishibata

Kobayashi

Sakai

Imagaki

Ohtani

Sakai

Yoshizumi

MK-1775, a small molecule Wee1 inhibitor, enhances anti-tumor efficacy of various DNA-damaging agents, including 5-fluorouracil

Cancer Biol Ther95145222010

10.4161/cbt.9.7.11115

20107315

Wang

Niu

Zhang

Caldwell

Edwards

Chen

Taub

Zhao

Synergistic antitumor interactions between MK-1775 and panobinostat in preclinical models of pancreatic cancer

Cancer Lett3566566682015

10.1016/j.canlet.2014.10.015

25458954

Ghiasi

Habibagahi

Rosli

Ghaderi

Yusoff

Hosseini

Abdullah

Jaberipour

Tumour suppressive effects of WEE1 gene silencing in breast cancer cells

Asian Pac J Cancer Prev14660566112014

10.7314/APJCP.2013.14.11.6605

24377575

Leijen

Beijnen

Schellens

Abrogation of the G2 checkpoint by inhibition of Wee-1 kinase results in sensitization of p53-deficient tumor cells to DNA-damaging agents

Curr Clin Pharmacol51861912010

10.2174/157488410791498824

20406171

Hirai

Iwasawa

Okada

Arai

Nishibata

Kobayashi

Kimura

Kaneko

Ohtani

Yamanaka

Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents

Mol Cancer Ther8299230002009

10.1158/1535-7163.MCT-09-0463

19887545

Rajeshkumar

De Oliveira

Ottenhof

Watters

Brooks

Demuth

Shumway

Mizuarai

Hirai

Maitra

Hidalgo

MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts

Clin Cancer Res17279928062011

10.1158/1078-0432.CCR-10-2580

21389100

Harris

Venkataraman

Alimova

Birks

Balakrishnan

Cristiano

Donson

Dubuc

Taylor

Foreman

Integrated genomic analysis identifies the mitotic checkpoint kinase WEE1 as a novel therapeutic target in medulloblastoma

Mol Cancer13722014

10.1186/1476-4598-13-72

24661910

Holzhauser

Lukoseviciute

Andonova

Ursu

Dalianis

Wickström

Kostopoulou

Targeting fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) signaling pathways in medulloblastoma cell lines

Anticancer Res4053662020

10.21873/anticanres.13925

31892552

Lukoseviciute

Maier

Poulou-Sidiropoulou

Rosendahl

Holzhauser

Dalianis

Kostopoulou

Targeting PI3K, FGFR, CDK4/6 signaling pathways together with cytostatics and radiotherapy in two medulloblastoma cell lines

Front Oncol117486572021

10.3389/fonc.2021.748657

34631586

Kostopoulou

Holzhauser

Lange

BKA

Ohmayer

Andonova

Bersani

Wickström

Dalianis

Analyses of FGFR3 and PIK3CA mutations in neuroblastomas and the effects of the corresponding inhibitors on neuroblastoma cell lines

Int J Oncol55137213842019

31638167

Zheng

Wang

Aldahdooh

Malyutina

Shadbahr

Tanoli

Pessia

Jing

SynergyFinder Plus: Toward better interpretation and annotation of drug combination screening datasets

Genomics Proteomics Bioinformatics205875962022

10.1016/j.gpb.2022.01.004

35085776

Smith

Hampton

Reynolds

Kang

Maris

Gorlick

Kolb

Lock

Carol

Keir

Initial testing (stage 1) of the PARP inhibitor BMN 673 by the pediatric preclinical testing program: PALB2 mutation predicts exceptional in vivo response to BMN 673

Pediatr Blood Cancer6291982015

10.1002/pbc.25201

25263539

Moreira

Venkataraman

Subramanian

Desisto

Balakrishnan

Prince

Pierce

Griesinger

Green

Eberhardt

Targeting MYC-driven replication stress in medulloblastoma with AZD1775 and gemcitabine

J Neurooncol1475315452020

10.1007/s11060-020-03457-0

32180106

Kreahling

Gemmer

Reed

Letson

Bui

Altiok

MK1775, a selective Wee1 inhibitor, shows single-agent antitumor activity against sarcoma cells

Mol Cancer Ther111741822012

10.1158/1535-7163.MCT-11-0529

22084170

Guertin

Liu

Hurd

Schuller

Long

Hirsch

Feldman

Benita

Toniatti

Preclinical evaluation of the WEE1 inhibitor MK-1775 as single-agent anticancer therapy

Mol Cancer Ther12144214522013

10.1158/1535-7163.MCT-13-0025

23699655

Byskata

Lukoseviciute

Tuti

Zupancic

Kostopoulou

Holzhauser

Dalianis

Targeted therapy with PI3K, PARP, and WEE1 inhibitors and radiotherapy in HPV positive and negative tonsillar squamous cell carcinoma cell lines reveals synergy while effects with APR-246 are limited

Cancers (Basel)15932022

10.3390/cancers15010093

36612094

Heijink

Blomen

Bisteau

Degener

Matsushita

Kaldis

Foijer

van Vugt

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

Proc Natl Acad Sci USA11215160151652015

10.1073/pnas.1505283112

26598692

Guney Eskiler

Cecener

Egeli

Tunca

Synthetically lethal BMN 673 (talazoparib) loaded solid lipid nanoparticles for BRCA1 mutant triple negative breast cancer

Pharm Res352182018

10.1007/s11095-018-2502-6

30255456

Bucher

Britten

G2 checkpoint abrogation and checkpoint kinase-1 targeting in the treatment of cancer

Br J Cancer985235282008

10.1038/sj.bjc.6604208

18231106

Figure 1.

Effects of WEE1 (MK-1775) and PARP (BMN673) single and combination treatments on the viability of DAOY, UW228-3 (SHH group), D425 and MED8A (group 3) medulloblastoma cell lines. The absorbance was measured 24, 48 and 72 h following the addition of (A-D) MK-1775, (E-H) BMN673 or (I-L) their combination. The graphs represent at least three experimental runs per cell line. MK, MK-1775; BMN, BMN673.

Figure 2.

Representation of (A-D) 2D and (E-H) 3D surface plots illustrating the optimal concentrations of MK-1775 and BMN673 in medulloblastoma cell lines. The synergy score was calculated using the Synergy Finder plus program. HSA values >10 indicated synergistic effects of the drugs, HSA values from −10 to 10 indicated additive effects, and HSA values <-10 indicated antagonism. MK, MK-1775; BMN, BMN673; HSA, highest single agent.

Figure 3.

Effects of with WEE1 (MK-1775) and PARP (BMN673) inhibitors alone and in combination on the confluency of DAOY and UW228-3 medulloblastoma cell lines. The cells were followed for up to 72 h after treatment. Single treatments are presented for (A and D) MK-1775 and for (B and E) BMN673. (C and F) Combined treatments are presented. MK, MK-1775; BMN, BMN673.

Figure 4.

Cytotoxic effects of WEE1 (MK-1775) and PARP (BMN673) inhibitors alone and in combination on DAOY and UW228-3 medulloblastoma cell lines. The cytotoxic effects of the inhibitors on the DAOY and UW228-3 cells were measured for up to 72 h after treatment. Single treatments are presented for (A and D) MK-1775 and for (B and E) BMN673. (C and F) Combined treatments are presented. The graphs represent one experimental run per cell line. MK, MK-1775; BMN, BMN673.

Figure 5.

Cell cycle profiles and analysis of each cell cycle phase of DAOY and UW228-3 MB cell lines following treatment with WEE1 (MK-1775) and PARP (BMN673) inhibitors alone or in combination for 48 h. Representative cell cycle profiles of (A) DAOY and (B) UW228-3 MB cell lines are shown on the upper panel and the percentages of cell distribution in the different cell cycle phases are presented on the bottom panel. The percentages represent three experimental runs per cell line. Ctl, control; BMN, BMN673; MK, MK-1775, MB, medulloblastoma.