Potentiating radiotherapy and chemotherapy by inhibiting DNA damage repair is proposed as a therapeutic strategy to improve outcomes for patients with solid tumors. However, this approach risks enhancing normal tissue toxicity as much as tumor toxicity, thereby limiting its translational impact. Using NU5455, a newly identified highly selective oral inhibitor of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity, we found that it was indeed possible to preferentially augment the effect of targeted radiotherapy on human orthotopic lung tumors without influencing acute DNA damage or a late radiation-induced toxicity (fibrosis) to normal mouse lung. Furthermore, while NU5455 administration increased both the efficacy and the toxicity of a parenterally administered topoisomerase inhibitor, it enhanced the activity of doxorubicin released locally in liver tumor xenografts without inducing any adverse effect. This strategy is particularly relevant to hepatocellular cancer, which is treated clinically with localized drug-eluting beads and for which DNA-PKcs activity is reported to confer resistance to treatment. We conclude that transient pharmacological inhibition of DNA-PKcs activity is effective and tolerable when combined with localized DNA-damaging therapies and thus has promising clinical potential.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8⁺ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell–specific functions. C5aR1 targeting resulted in increased NF-κB–dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.

Due to the lack of effective therapies and poor prognosis in TNBC (triple-negative breast cancer) patients, there is a strong need to develop effective novel targeted therapies for this subtype of breast cancer. Inhibition of heat shock protein 90 (HSP90), a conserved molecular chaperone that is involved in the regulation of oncogenic client proteins, has shown to be a promising therapeutic approach for TNBC. However, both intrinsic and acquired resistance to HSP90 inhibitors (HSP90i) limits their effectiveness in cancer patients. We developed models of acquired resistance to HSP90i by prolonged exposure of TNBC cells to HSP90i (ganetespib) in vitro. Whole transcriptome profiling and a 328-compound bioactive small molecule screen were performed on these cells to identify the molecular basis of acquired resistance to HSP90i and potential therapeutic approaches to overcome resistance. Among a panel of seven TNBC cell lines, the most sensitive cell line (Hs578T) to HSP90i was selected as an in vitro model to investigate acquired resistance to HSP90i. Two independent HSP90i-resistant clones were successfully isolated which both showed absence of client proteins degradation, apoptosis induction and G2/M cell cycle arrest after treatment with HSP90i. Gene expression profiling and pathway enrichment analysis demonstrate significant activation of the survival JAK-STAT signalling pathway in both HSP90i-resistant clones, possibly through IL6 autocrine signalling. A bioactive small molecule screen also demonstrated that the HSP90i-resistant clones showed selective sensitivity to JAK2 inhibition. Inhibition of JAK and HSP90 caused higher induction of apoptosis, despite prior acquired resistance to HSP90i. Acquired resistance to HSP90i in TNBC cells is associated with an upregulated JAK-STAT signalling pathway. A combined inhibition of the JAK-STAT signalling pathway and HSP90 could overcome this resistance. The benefits of the combined therapy could be explored further for the development of effective targeted therapy in TNBC patients.

We recently reported that genetic or pharmacological inhibition of insulin-like growth factor receptor (IGF-1R) slows DNA replication and induces replication stress by downregulating the regulatory subunit RRM2 of ribonucleotide reductase, perturbing deoxynucleotide triphosphate (dNTP) supply. Aiming to exploit this effect in therapy we performed a compound screen in five breast cancer cell lines with IGF neutralising antibody xentuzumab. Inhibitor of checkpoint kinase CHK1 was identified as a top screen hit. Co-inhibition of IGF and CHK1 caused synergistic suppression of cell viability, cell survival and tumour growth in 2D cell culture, 3D spheroid cultures and in vivo. Investigating the mechanism of synthetic lethality, we reveal that CHK1 inhibition in IGF-1R depleted or inhibited cells further downregulated RRM2, reduced dNTP supply and profoundly delayed replication fork progression. These effects resulted in significant accumulation of unreplicated single-stranded DNA and increased cell death, indicative of replication catastrophe. Similar phenotypes were induced by IGF:WEE1 co-inhibition, also via exacerbation of RRM2 downregulation. Exogenous RRM2 expression rescued hallmarks of replication stress induced by co-inhibiting IGF with CHK1 or WEE1, identifying RRM2 as a critical target of the functional IGF:CHK1 and IGF:WEE1 interactions. These data identify novel therapeutic vulnerabilities and may inform future trials of IGF inhibitory drugs.

Radiation-induced DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) and nonhomologous end joining (NHEJ). Recently, it has been found that chronic tumor hypoxia compromises HR repair of DNA DSBs but activates the NHEJ protein DNAPK. We therefore hypothesized that inhibition of DNAPK can preferentially potentiate the sensitivity of chronically hypoxic cancer cells to radiation through contextual synthetic lethality in vivo. In this study, we investigated the impact of DNAPK inhibition by a novel selective DNAPK inhibitor, NU5455, on the repair of radiation-induced DNA DSBs in chronically hypoxic and nonhypoxic cells across a range of xenograft models. We found that NU5455 inhibited DSB repair following radiation in both chronically hypoxic and nonhypoxic tumor cells. Most importantly, the inhibitory effect was more pronounced in chronically hypoxic tumor cells than in nonhypoxic tumor cells. This is the first in vivo study to indicate that DNAPK inhibition may preferentially sensitize chronically hypoxic tumor cells to radiotherapy, suggesting a broader therapeutic window for transient DNAPK inhibition combined with radiotherapy.