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p53-Mediated Radiosensitization of 177Lu-DOTATATE in Neuroblastoma Tumor Spheroids
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.ORCID iD: 0000-0002-0063-3233
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Preparative Medicinal Chemistry.ORCID iD: 0000-0001-9943-5976
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Neuro-Oncology.ORCID iD: 0000-0001-6458-1949
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2021 (English)In: Biomolecules, E-ISSN 2218-273X, Vol. 11, no 11, article id 1695Article in journal (Refereed) Published
Abstract [en]

p53 is involved in DNA damage response and is an exciting target for radiosensitization in cancer. Targeted radionuclide therapy against somatostatin receptors with 177Lu-DOTATATE is currently being explored as a treatment for neuroblastoma. The aim of this study was to investigate the novel p53-stabilizing peptide VIP116 in neuroblastoma, both as monotherapy and together with 177Lu-DOTATATE. Five neuroblastoma cell lines, including two patient-derived xenograft (PDX) lines, were characterized in monolayer cultures. Four out of five were positive for 177Lu-DOTATATE uptake. IC50 values after VIP116 treatments correlated with p53 status, ranging between 2.8–238.2 μM. IMR-32 and PDX lines LU-NB-1 and LU-NB-2 were then cultured as multicellular tumor spheroids and treated with 177Lu-DOTATATE and/or VIP116. Spheroid growth was inhibited in all spheroid models for all treatment modalities. The most pronounced effects were observed for combination treatments, mediating synergistic effects in the IMR-32 model. VIP116 and combination treatment increased p53 levels with subsequent induction of p21, Bax and cleaved caspase 3. Combination treatment resulted in a 14-fold and 1.6-fold induction of MDM2 in LU-NB-2 and IMR-32 spheroids, respectively. This, together with differential MYCN signaling, may explain the varying degree of synergy. In conclusion, VIP116 inhibited neuroblastoma cell growth, potentiated 177Lu-DOTATATE treatment and could, therefore, be a feasible treatment option for neuroblastoma.

Place, publisher, year, edition, pages
MDPI, 2021. Vol. 11, no 11, article id 1695
Keywords [en]
neuroblastoma, radionuclide therapy, p53, 177Lu-DOTATATE, radiosensitization, stapled peptides
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-459959DOI: 10.3390/biom11111695ISI: 000724965100001PubMedID: 34827693OAI: oai:DiVA.org:uu-459959DiVA, id: diva2:1615651
Funder
Swedish Research Council, 2020-01377Swedish Cancer Society, CAN 2018/494Swedish Cancer Society, CAN 20 0191Swedish Childhood Cancer Foundation, PR2020-0023Swedish Childhood Cancer Foundation, TJ2021-0072
Note

Title in Web of Science: p53-Mediated Radiosensitization of Lu-177-DOTATATE in Neuroblastoma Tumor Spheroids

Available from: 2021-11-30 Created: 2021-11-30 Last updated: 2024-12-06Bibliographically approved
In thesis
1. Searching for Synergy: Radiosensitization of 177Lu-DOTATATE
Open this publication in new window or tab >>Searching for Synergy: Radiosensitization of 177Lu-DOTATATE
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cancers presents a major health challenge, and there is a pressing need to develop new therapeutic strategies. Surgery, chemotherapy and radiation are the most commonly used treatments for cancer today. Radiation can be given as targeted radionuclide therapy (TRT), i.e., systemic administration of a radiolabeled cancer-targeting molecule. This is especially suitable for inoperable and disseminated tumors.

177Lu-DOTATATE, a TRT directed against the somatostatin receptors (SSTRs), was recently approved for therapy of a subset of neuroendocrine tumors (NETs). Although it has prolonged the life of NET patients, complete remission is seldom achieved. Consequently, to increase the efficacy of the treatment, this thesis aimed to assess potential radiosensitizing strategies for 177Lu-DOTATATE. The two radiosensitization targets in focus were HSP90, a chaperone protein with numerous oncogenic client proteins, and p53, a central regulator of DNA damage.

In papers I and II, we investigated the HSP90-inhibitor Onalespib, as a treatment for NETs, and as a potential radiosensitizer. The drugs were assessed in vitro and in vivo. We concluded that Onalespib reduced NET cell growth and acted synergistically with 177Lu-DOTATATE. Inhibition of EGFR, a HSP90 client protein, was suggested as a mediator of the observed synergy. Furthermore, the combination had a favorable toxicity profile. 

In paper III, we assessed the novel stapled peptide VIP116, which inhibits the p53 repressors MDM2 and MDM4, as a potentiator of 177Lu-DOTATATE in wildtype p53 neuroblastoma cells. Combination therapy exhibited growth-inhibitory effects, with resulting additive or synergistic effects. The treatment-mediated effects on p53 signaling were characterized, revealing a possible involvement of V-myc myelocytomatosis viral oncogene homolog, neuroblastoma derived (MYCN), a prognostic marker for poor survival in neuroblastoma.

In paper IV, we aimed to improve targeted delivery of VIP116, with the use of lipid bilayer disks (lipodisks). VIP116 was successfully loaded onto epidermal growth factor receptor (EGFR)-targeting lipodisks, leading to specific delivery and reduction of viability of EGFR expressing tumor cells. The study provided a proof-of-concept for utilizing lipodisks as a drug delivery system for p53-stabilizing peptides.

In conclusion, we have investigated, and found, suitable candidates for potentiating 177Lu-DOTATATE therapy. We have addressed the feasibility of the treatments, toxicity and targeted delivery. Moreover, the work has explored the biology of TRT. This is an area in need of more attention, as more and more radionuclide-based therapies are entering clinicals trials and reaching approval.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2022. p. 58
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1797
Keywords
Cancer, targeted radionuclide therapy, radiosensitization, p53, MDM2/MDM4 inhibition, HSP90, drug synergy, drug delivery, lipid nanoparticles, lipodisks
National Category
Cell and Molecular Biology Cancer and Oncology
Research subject
Biomedical Radiation Science
Identifiers
urn:nbn:se:uu:diva-459961 (URN)978-91-513-1358-0 (ISBN)
Public defence
2022-02-04, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Funder
Swedish Cancer Society, 2018/494Swedish Cancer Society, 2016/649Swedish Cancer Society, 2015/1080Swedish Cancer Society, 2015/385Swedish Research Council, 2013-30876-104113-30Swedish Childhood Cancer Foundation, PR3018-0067Clas Groschinski Memorial FoundationSwedish Childhood Cancer Foundation, PR2020-0023Swedish Childhood Cancer Foundation, TJ2021-0072Swedish Research Council, 2020-01377Swedish Cancer Society, CAN 20 0191Swedish Cancer Society, CAN 2017/422
Available from: 2022-01-10 Created: 2021-12-01 Last updated: 2022-01-18
2. To a Radiant Future and Beyond: Improving Radiotherapy of Neuroblastoma
Open this publication in new window or tab >>To a Radiant Future and Beyond: Improving Radiotherapy of Neuroblastoma
2025 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Neuroblastoma is a pediatric cancer with a five-year survival rate of merely 50% for high-risk cases. The treatment regimen is aggressive, leading to extensive side effects that significantly impact patients’ quality of life.

Targeted radionuclide therapy (TRT) involves the systemic administration of cancer-specific radioconjugates. This thesis focuses on TRT against the somatostatin receptor 2 (SSTR2) and the antigen CD44v6, two targets that are overexpressed in neuroblastoma,  

Radiosensitization renders cells more sensitive to radiation, which can improve the therapeutic efficacy and potentially reduce the radiation dose required to achieve an antitumor effect. This thesis investigates radiosensitization through the stabilization of p53 and the inhibition of heat shock protein 90 (HSP90), two proteins involved in the cellular response to DNA damage.

In papers I and II, we investigated the combination of the SSTR2-targeting radioconjugate 177Lu-DOTATATE with the p53-stabilizing peptide VIP116 for neuroblastoma treatment. The combination therapy demonstrated enhanced antitumor effects in both in vitro and in vivo studies using mice bearing human neuroblastoma xenografts. Notably, the untreated and monotreated controls showed no nephrotoxicity.

In paper III, we demonstrated that combining external beam radiotherapy with the HSP90-inhibitor Onalespib produced additive or synergistic effects in vitro across a panel of neuroblastoma cell lines. Additionally, mice bearing syngeneic neuroblastoma tumor xenografts treated with this combination exhibited significantly improved therapeutic efficacy compared to control groups.

In paper IV, we developed and characterized human anti-CD44v6 antibodies for molecular radiotherapy. This work identified a lead candidate, UU-40, which demonstrated high affinity, strong tumor uptake, and favorable in vivodistribution, making it a promising candidate for future use against CD44v6-expressing cancers.

In conclusion, this thesis demonstrates that radiosensitization enhances the antitumor effects of radiation therapy in preclinical models of neuroblastoma. It is our hope that these discoveries will enable more effective and less harmful treatments for children with neuroblastoma. This thesis also produced an anti-CD44v6 antibody that holds great potential for future use in targeted radionuclide therapy, paving the way for innovative treatments for CD44v6-expressing cancers, including neuroblastoma. 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2025. p. 53
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 2112
Keywords
Cancer, targeted radionuclide therapy, external beam radiotherapy, radiosensitization, neuroblastoma, p53, MDM2/MDM4 inhibition, HSP90, CD44v6, antibodies
National Category
Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-544449 (URN)978-91-513-2330-5 (ISBN)
Public defence
2025-02-07, Rudbecksalen, Rudbeck laboratory, Dag Hammarskjölds Väg 20, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2025-01-13 Created: 2024-12-06 Last updated: 2025-01-13

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Lundsten, SaraBerglund, HannaJha, PreetiKrona, CeciliaHariri, MehranNelander, SvenLane, David P.Nestor, Marika

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