Novel strategies of optimal combinations of targeted therapy in medulloblastoma and neuroblastoma

<p dir="ltr"><b>Background/Aim</b>: Medulloblastoma (MB) and neuroblastoma (NB) are among the most common malignant solid tumors in children. Despite decades of clinical advances, these cancers often remain difficult to treat due to their heterogeneous and aggressive nature, the emergence of treatment resistance and the toxicity associated with standard therapies such as chemotherapy and radiotherapy. While current multimodal treatments succeed in improving survival in some patient groups, they often result in significant long-term side effects. In addition, relapse and refractory disease continue to be major clinical challenges, emphasizing the urgent need for more effective and less toxic therapeutic strategies. Targeted therapies, which selectively inhibit key oncogenic drivers and molecular pathways involved in tumor progression, offer a promising alternative to traditional treatments. In this PHD thesis, we investigated the preclinical efficacy of mostly FDA-approved inhibitors of PI3K, FGFR, CDK4/6, PARP, WEE1 and AKT, alone or in various combinations, on both MB and NB models using two-dimensional (2D) and 3D cell culture settings.</p><p dir="ltr"><b>Material and methods</b>: MB cell lines (DAOY, UW228-3, D425, Med8A, and D283) and NB cell lines (SK-N-AS, SK-N-BE(2)-C, SK-N-FI, SK-N-DZ, and SK-N-SH) were cultured and treated with inhibitors against PI3K (BYL719), FGFR (JNJ- 42756493), CDK4/6 (PD-0332991), AKT (AZD5363), PARP (BMN673) and WEE1 (MK-1775). The inhibitors were evaluated both as monotherapies and in various combinations with each other, as well as in combination with conventional chemotherapy and radiotherapy. Their effects were assessed on cell viability, growth, proliferation, cytotoxicity, apoptosis, cell cycle distribution and migration in both 2D and 3D culture models.</p><p dir="ltr"><b>Key findings</b>: In MB, most drugs presented dose-dependent effects. Moreover, the PI3K inhibitor BYL719 demonstrated synergistic anti-tumor activity when combined with the CDK4/6 inhibitor PD-0332991, the FGFR inhibitor JNJ- 42756493, as well as with the chemotherapeutic agents cisplatin and vincristine. The efficacy of these single inhibitors was further enhanced when combined with radiotherapy. Notably, the WEE1 inhibitor MK-1775, either alone or in combination with the PARP inhibitor BMN673, was effective specifically in Sonic Hedgehog (SHH)-subgroup cell lines (DAOY and UW228-3), but not in Group 3 cell lines (Med8A and D425). In addition, the AKT inhibitor AZD5363 showed synergistic effects when combined with BYL719, cisplatin or vincristine across several MB cell lines.</p><p dir="ltr">In NB, most drugs presented dose-dependent effects. Moreover, the combination of PI3K and FGFR inhibitors led to enhanced anti-tumor efficacy. However, when paired with chemotherapeutic agents such as cisplatin, vincristine or doxorubicin, the effects varied among NB cell lines, ranging from synergistic to antagonistic responses. Notably, combining the PI3K inhibitor BYL719 and the CDK4/6 inhibitor PD-0332991 resulted in consistent synergistic effects across all NB cell lines. Similarly, combining the PARP inhibitor BMN673 with the WEE1 inhibitor MK-1775 produced additive or synergistic responses in most NB cell lines, although BMN673 alone was largely ineffective.</p><p dir="ltr"><b>Conclusions</b>: This thesis provides compelling pre-clinical evidence supporting the therapeutic potential of repurposing and combining targeted inhibitor combinations in MB and NB. In MB, PI3K, FGFR and CDK4/6 inhibitors in combination with each other or with radiotherapy demonstrated promising efficacy. In NB, PI3K inhibitors paired with FGFR or CDK4/6 inhibitors also showed strong anti-tumor activity. However, combining these inhibitors with standard chemotherapeutics produced variable responses in both MB and NB, highlighting the need for further investigation. Notably, the combination of WEE1 and PARP inhibitors was effective in SHH MB and NB, whereas PARP inhibition alone showed limited efficacy. Finally, AKT inhibitor showed synergistic interactions when combined with PI3K inhibitors or chemotherapy in MB. By identifying synergistic interactions across both simple 2D and advanced 3D models, this work lays the foundation for the development of more precise and less toxic treatment strategies. Crucially, the findings suggest that rational, mechanism-driven drug combinations may not only enhance survival outcomes but also help preserve the quality of life in pediatric cancer patients. Overall, this thesis contributes to the growing body of evidence supporting the shift towards personalized and mechanism-based treatment therapies in pediatric oncology.</p><h3>List of scientific papers</h3><p dir="ltr">I. <b>Lukoseviciute M,</b> Maier H, Poulou-Sidiropoulou E, Rosendahl E, Holzhauser S, Dalianis T, Kostopoulou ON. Targeting PI3K, FGFR, CDK4/6 Signaling Pathways Together With Cytostatics and Radiotherapy in Two Medulloblastoma Cell Lines. Front Oncol. 2021 Sep 24;11:748657. <a href="https://doi.org/10.3389/fonc.2021.748657" target="_blank">https://doi.org/10.3389/fonc.2021.748657</a></p><p dir="ltr">II. <b>Lukoseviciute M,</b> Need E, Birgersson M, Dalianis T, Kostopoulou ON. Enhancing targeted therapy by combining PI3K and AKT inhibitors with or without cisplatin or vincristine in medulloblastoma cell lines in vitro. Biomed Pharmacother. 2024 Nov;180:117500. <a href="https://doi.org/10.1016/j.biopha.2024.117500" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.biopha.2024.117500</a></p><p dir="ltr">III. <b>Lukoseviciute M,</b> Theodosopoulou A, Holzhauser S, Dalianis T, Kostopoulou ON. Combination of PARP and WEE1 inhibitors in vitro: Potential for use in the treatment of SHH medulloblastoma. Oncol Rep. 2023 Jun;49(6):125. <a href="https://doi.org/10.3892/or.2023.8562" rel="noreferrer" target="_blank">https://doi.org/10.3892/or.2023.8562</a></p><p dir="ltr">IV. Holzhauser S, <b>Lukoseviciute M,</b> Papachristofi C, Vasilopoulou C, Herold N, Wickström M, Kostopoulou ON*, Dalianis T *. Effects of PI3K and FGFR inhibitors alone and in combination, and with/without cytostatics in childhood neuroblastoma cell lines. Int J Oncol. 2021 Feb;58(2):211-225.<a href=" https://doi.org/10.3892/ijo.2021.5167" rel="noreferrer" target="_blank"> https://doi.org/10.3892/ijo.2021.5167</a></p><p dir="ltr">V. <b>Lukoseviciute M,</b> Holzhauser S, Pappa E, Mandal T, Dalianis T, Kostopoulou ON. Efficacy of combined targeted therapy with PI3K and CDK4/6 or PARP and WEE1 inhibitors in neuroblastoma cell lines. Oncol Rep. 2023 Sep;50(3):166. <a href="https://doi.org/10.3892/or.2023.8603" rel="noreferrer" target="_blank">https://doi.org/10.3892/or.2023.8603</a></p><p dir="ltr">VI. <b>Lukoseviciute M,</b> Need E, Holzhauser S, Dalianis T, Kostopoulou ON. Combined targeted therapy with PI3K and CDK4/6, or FGFR inhibitors show synergistic effects in a neuroblastoma spheroid culture model. Biomed Pharmacother. 2024 Aug;177:116993. <a href="https://doi.org/10.1016/j.biopha.2024.116993" rel="noreferrer noopener" target="_blank">https://doi.org/10.1016/j.biopha.2024.116993</a></p>