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Cancer, a complex disease marked by uncontrolled cell growth, is typically treated with surgery, chemotherapy, radiation therapy and immunotherapy, which can induce significant side effects by affecting healthy tissues. Targeted radionuclide therapy (TRT), where cancer-targeting molecules are equipped with radionuclides to enable cancer-specific radiotherapy, shows promise for treating advanced cancers by addressing both metastatic relapse and heterogeneous tumors. Combining TRT with targeted therapies offers a promising shift towards more effective and less toxic treatments. This thesis focuses on synergistically enhancing the therapeutic efficacy of TRT or chemotherapy through combination strategies with novel drugs that modulate DNA damage and/or interact with the immune system.

In Paper I, we investigated the combination treatment of the chemotherapy drug cisplatin with the heat shock protein 90 (HSP90) inhibitor onalespib in vitro, using ovarian and head and neck cancer cells. Our findings demonstrated that onalespib enhances the therapeutic effects of cisplatin, reducing colony formation and migration, increasing apoptosis, and decreasing DNA damage response (DDR). Key proteins such as ATM, DNA-PKcs, and γH2AX were shown to play crucial roles in the therapeutic efficacy of the combination treatments.

In Papers II and III, we characterized the synergy between the novel radioconjugate, ¹⁷⁷Lu-DOTA-M5A, and onalespib in gastrointestinal cancer models in vitro and in vivo. While ¹⁷⁷Lu-DOTA-M5A exhibited significant cellular uptake and therapeutic efficacy as monotherapy in 3D tumor spheroids and xenografts. The combination exhibited the most pronounced synergistic growth inhibitory effects in both settings with no adverse effects observed in vivo. PARP1 was identified as playing a pivotal role in the therapeutic outcomes.

In Paper IV, we explored combining ¹⁷⁷Lu-DOTA-M5A with PD-1 immune checkpoint blockade in an immunocompetent transgenic mouse model. The radioconjugate demonstrated high tumor uptake and potent therapeutic effects as monotherapy without depleting immune cells within the tumor microenvironment, while PD-1 blockade further enhanced its efficacy by prolonging survival and suppressing tumor growth. CD8⁺ T cells and pro-inflammatory macrophages (M1) were critical for these therapeutic effects and no myelotoxicity was observed with any treatments.

In conclusion, we have investigated various combination treatment approaches aimed at enhancing therapeutic efficacy while mitigating side effects and drug resistance. We have evaluated the feasibility, toxicity, and benefits of these combinations in preclinical settings with promising results, underscoring the potential of integrating TRT into combination therapy.

Further investigation is warranted as an increasing number of TRT and combination therapies are entering clinical trials.

Human DNA constantly faces endogenous and exogenous damage, with DNA double-strand breaks (DSBs) posing the greatest threat to genome integrity. However, DSBs can be leveraged to kill cancer cells, as many treatments act as DSB inducers. The dominant repair pathway, non-homologous end-joining (NHEJ), resolves the majority of DSBs. This thesis explores strategies to sensitize resistant cancer cells through combination therapy and investigates NHEJ’s response to varying DSB complexities.

Paper I addresses cisplatin resistance in ovarian cancer. We found that combining cisplatin with the HSP90 inhibitor onalespib enhances sensitivity by increasing DSB levels, inducing apoptosis, and causing G2/M arrest, making it a promising strategy. Paper II focuses on glioblastoma (GBM), an aggressive brain tumor with limited treatment options. We demonstrated that onalespib enhances radiosensitivity in 2D and 3D GBM models by increasing DSB levels, promoting apoptosis, and altering protein expression, suggesting that HSP90 inhibition could improve radiotherapy outcomes. Paper III investigates the alpha emitter Ra-223, used in bone-metastatic prostate cancer. Our findings revealed that Ra-223 generates clustered DSBs, triggering NHEJ activation, growth inhibition, and apoptosis in prostate cancer cells, with no detectable cellular uptake. Paper IV explores pharmacological ascorbate (Asc) effect on NHEJ pathway. We found that Asc induces delayed DSBs, extensive pan-nuclear γH2AX formation, necrosis, and G2/M arrest in colorectal cancer cells, with stronger effects in XRCC4 KO cells. We concluded that Asc does not generate prompt DSBs, and the delayed DSBs are linked to necrotic nuclear degradation, with sensitivity influenced by cell cycle regulation rather than NHEJ deficiency. Paper V examines NHEJ’s role in repairing DSBs of varying complexity in colorectal cancer cells. Wild-type cells exhibited both fast and slow repair kinetics, while NHEJ-deficient cells showed only a fast repair phase, followed by repair failure. Non-DSB clusters increased as the DSB:SSB ratio decreased (from calicheamicin to X-rays, bleomycin, etoposide, and temozolomide). These clusters were rapidly removed, independent of NHEJ, highlighting the impact of DSB type/complexity on repair efficiency.

In conclusion, this thesis presents strategies to overcome cisplatin resistance, enhance radiosensitivity in GBM, and elucidate Ra-223 toxicity mechanisms in prostate cancer. It also examines Asc’s effects on DSB induction and repair and reveals NHEJ’s role in processing complex DSBs. Our findings provide new insights into optimizing DSB repair and therapeutic strategies in cancer treatment.