Date Available

5-15-2024

Year of Publication

2024

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Clinical and Translational Science

First Advisor

Dr. B. Mark Evers

Abstract

Gastroenteropancreatic Neuroendocrine Tumor (GEP-NET) is a heterogeneous group of malignancies arising from multipotent neuroendocrine stem cells in the gastrointestinal tract. The incidence of GEP-NET continues to rise, possibly due to the advancement of imaging and biomarkers available for diagnosis. However, even though the majority of patients are diagnosed with low-grade and localized disease, a significant percentage of patients present with advanced-stage metastatic disease with poor prognosis in months to a few years. These advanced-stage GEP-NET patients can also present with a variety of debilitating symptoms that significantly impact their quality of life. The role of palliative-intent surgery is limited, and systemic treatment is the mainstay for treating advanced-stage metastatic GEP-NETs. The systemic treatment options include long-acting somatostatin analogs, telotristat ethyl, interferon alpha, anti-angiogenesis agents, immunotherapy, targeted therapy, and conventional chemotherapy.

Radiotherapy is an emerging modality in managing advanced-stage metastatic GEP-NET. The most common type of radiotherapy is the external beam radiation (EBRT), which has been implemented as a palliative measure with reasonable locoregional control. Another novel modality is the Y-90 radioembolization, which is a liver-directed therapy for GEP-NET patients with hepatic metastases. The most widely recognized novel treatment in the modern era is the peptide radionuclide receptor therapy (PRRT). In landmark clinical trials, PRRT has demonstrated an improved objective response rate compared to the standard of care long-acting somatostatin analog systemic treatment. Therefore, it has emerged as the new standard of care in managing advanced-stage metastatic GEP-NETs. Despite its promising outcomes, PRRT failed to demonstrate improved survival, and the objective response rate was still considered suboptimal. One area of active research is the development of combined therapy with PRRT to enhance treatment efficacy, and ultimately improve patient outcomes. Only three anti-neoplastic agents with radiosensitizing property have been established in existing clinical trials, though several therapeutic agents are currently under investigations. This thesis aims to investigate novel radiosensitizing therapeutic agents in preclinical studies and demonstrate their translational values for prospective clinical trials.

The mammalian-targeted rapamycin receptor (mTOR) dysregulation has been established as a crucial therapeutic target for GEP-NET. A study investigated the radiosensitization of PI3K/mTOR dual inhibitor in GEP-NET cell lines (QGP-1, BON, NT-3) in vitro. We assessed the efficacy of PI3K/mTOR dual inhibitor PF-04691502 to inhibit pAkt and to increase apoptosis in GEP-NET cell lines and patient-derived tumor spheroids as a single agent or combined with radiotherapy. Treatment with PI3K/mTOR inhibitor decreased pAkt (Ser473) expression for up to 72h compared with control. Interestingly, simultaneous treatment with PI3K/mTOR dual inhibitor and X-ray ionizing radiotherapy did not induce significant apoptosis; however, the addition of PI3K/mTOR dual inhibitor 48h after radiotherapy significantly increased apoptosis compared to either PI3K/mTOR dual inhibitor or radiotherapy alone. This result demonstrated that the radiosensitization effect of PI3K/mTOR dual inhibitor is schedule-dependent. Our findings supported that radiotherapy in combination with appropriately scheduled PI3K/mTOR dual inhibitor may be a promising regimen for GEP-NET patients.

Another therapeutic target is the ribonucleotide reductase (RNR), a rate-limiting enzyme that produces deoxyribonucleoside triphosphates, building blocks for DNA synthesis and repair. We explored the radiosensitization effect by inhibiting RNR with a selective RNR M2 subunit (RRM2) inhibitor in metastatic pancreatic neuroendocrine tumor (pNET) cell lines (QGP-1 and BON) in vitro and in vivo. We found that RRM2 inhibition activated DNA damage response pathways by phosphorylating ATM and DNA-PKcs, but not ATR. RRM2 also increased G1 phase cell cycle arrest via Chk1 and Chk2 phosphorylation. The selective RRM2 inhibitor induced more apoptosis when combined with radiotherapy in vitro. We also utilized two metastatic pNET subcutaneous and lung metastasis models. We demonstrated significantly increased apoptosis of BON-cell subcutaneous xenograft and reduced lung metastases burden when combining selective RRM2 inhibitor with radiotherapy in vivo. Together, our findings successfully showed radiosensitization with selective RRM2 inhibitor in treating metastatic pNET and supported future clinical trials utilizing RRM2 inhibitor as a radiosensitizer in treating metastatic pNET.

In conclusion, our preclinical studies suggested that implementing radiosensitizer appropriately could induce more apoptosis, thus effectively reducing disease burden in multiple models in vitro and in vivo. The selection of a radiosensitizer should consider the unique genetics and molecular biomarkers of GEP-NET and proper radiobiology for radiosensitization. Several challenges of radiosensitizer research in GEP-NET include the relative rarity of disease that leads to a lack of preclinical models and slow recruitment in clinical trials, excessive toxicity with current generation radiosensitizers, and diverse tumor biology that renders the “one-fit-for-all” approach ineffective. In response to these challenges, we should consider leveraging modern technologies, such as artificial intelligence, to propel basic and translational GEP-NET research effectively. The future generation of an ideal radiosensitizer for treating GEP-NET is likely safe, effective, and personalized.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.177

Funding Information

This thesis was supported by the National Institute of Health T32 training grant CA 160003, and the Amanda W. Lockey Foundation at the Markey Cancer Center, Lexington, KY.

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