Multimodality combination strategies with chemotherapeutics might improve RT response while providing systemic control in treatment-resistant TNBC [14, 15]. Genomic instability is known to increase cancer cell radiosensitivity [16]. Exploring this vulnerability through combinations of RT and systemic agents is a promising strategy. PLK4 inhibition is an emerging new strategy for cancer, including breast cancer, and has been extensively studied [17, 18]. Various PLK4 inhibitors have been described, such as YLT11 [19] and Centrinone B [20]. However, CFI-400945 is the compound that has been the most extensively studied and has entered clinical trials (NCT03624543) in patients with breast cancer [5]. Mason et al. (2014) [3] characterized CFI-400945, investigating it as a single agent, and provided the foundational concepts regarding this compound. Given its promise as a novel anti-breast cancer agent and orally available drug, our group investigated if the combination of this compound with RT would enhance anticancer effects through further exacerbation of genomic instability [6]. In that study, the first to our knowledge to examine the combination of PLK4 inhibitors with RT, the synergistic effect of CFI-400945 and RT was shown. However, limited models were initially used to test the efficacy of this combination. Moreover, due to potential off-target effects of CFI-400945, it remained to be confirmed that the combination effect was indeed acting directly through PLK4 inhibition. The mechanistic effects of the combination treatment remained unclear. The latter has important translational implications in terms of studying other PLK4 inhibitors with RT to enhance anticancer effects. The aforementioned issues have been addressed in the current study.

Our previous [6] and current data provide strong support that CFI-400945 combined with RT works synergistically to deliver substantially greater anticancer effects than single agent treatments. Synergistic benefits of this combination were confirmed in translationally-relevant PDO models, suggesting that it might be effective in tackling cancer heterogeneity and treatment resistance. In the current manuscript, we provide new information regarding the combination of PLK4 inhibition together with RT.

First, we investigated various sequencing schedules of CFI-400945 with RT and their antiproliferative effects. We did not observe substantial differences in combinatorial effects under various treatment sequencing schedules, likely due to the short periods of culture under various treatment conditions in vitro.

Secondly, we, for the first time, show that specific PLK4 inhibition, at least in part, is responsible for enhanced anticancer effects previously observed upon combination treatment with CFI-400945 and RT. Since CFI-400945 is known to exhibit off-target effects at higher concentrations [4], we explored if PLK4 inhibition is in fact responsible for the antiproliferative synergistic effects with RT using a highly specific PLK4 inhibitor and RNA interference approaches. Reduction of PLK4 expression by siRNA or its inhibition with a highly-selective Centrinone B [4] also enhanced anticancer effects when combined with RT. Although combinatorial effects observed with CFI-400945 were overall stronger compared to siRNA silencing or Centrinone B treatment it is difficult to draw direct comparisons due to experimental differences, such as transient action of siRNA and drug concentrations. Overall, our data suggests that PLK4 is a promising target for enhancing RT effects in TNBC. These findings have important implications for translational research since other PLK4 inhibition strategies are being developed and might soon enter clinical trials [21]. Thus, the knowledge that the PLK4-specific inhibition is a promising strategy for enhancing effects of RT can be further utilized to investigate and develop multimodality treatment approaches in breast and potentially other cancers.

Next, we explored mechanistic aspects of the anticancer effects of PLK4 inhibition with RT. It is worth noting, that while PLK4 is known to play an essential role in centriole and centrosome control, the detailed mechanisms of its action are largely unknown. PLK4 has a known role in cell cycle control by regulating the process of centriole duplication [22]. Hence, inhibition of PLK4 can cause centrosome-related errors, such as centrosome amplification which leads to improper mitotic progression, genomic instability and subsequent cancer cell death [3]. While centriole overduplication is a purported mechanism of the anti-cancer effect of CFI-400945 as a single agent [3], other mechanisms might also be at play during its monotherapy or combination treatment. RT has been shown to induce centrosome amplification in cancer cells via centriole splitting or overamplification [23,24,25]. However, RT is also known to utilize a multitude of other mechanisms through which it asserts its anticancer effects [15]. Taken together, we aimed to investigate if the combination effect of CFI-400945 inhibition with RT works through centriole overduplication. We, for the first time, show that the combination effect of the drug and RT is, at least in part, promoted by centriole overduplication. The latter is indicative of genomic instability and mitotic catastrophe, which may lead to cancer cell death [24,25,26,27].

Our data suggest a model whereby the combined action of PLK4 inhibition and RT leads to increased overamplification of centrioles, which in turn increases genomic instability, compromises the ability of cancer cells to cope with genotoxic stress and results in enhanced anticancer effects. Additional studies might further shed light on the mechanism of action of PLK4 inhibition and RT, serve to identify novel targets for radiosensitization and facilitate translation of this approach to a clinical setting to improve outcomes in patients with TNBC.

Overall, our study shows that PLK4 inhibition together with RT, compared to single agent treatments, further enhances anticancer effects in TNBC. This novel multimodality approach can be potentially utilized in downstaging the primary and axillary lymph node tumors in the neoadjuvant setting, commonly used in TNBC, to improve operability or to convert inoperable tumors to operable ones. This approach might also be proven useful in the metastatic setting (e.g. skin, liver, bones) where radiotherapy effects on the metastatic deposit can be further enhanced by concurrent administration of CFI-400945. Similarly, the combination can be explored in the postoperative, or adjuvant setting to potentially decrease the incidence of locoregional recurrence. The fact that CFI-400945 is orally available makes it an attractive candidate for the multimodal treatment with RT in these settings. Notably, and pending results of Phase II (NCT03624543) trials for CFI-400945 as a single agent, its administration with RT might provide benefits of not only improved loco-regional disease control but also systemic disease control. Due to adverse effects, such as neutropenia, observed upon CFI-400945 treatment in Phase I trials [5], lower doses of the drug could be explored in clinical studies together with RT. Potential benefits of these various management approaches can be further explored in clinical trials with RT and CFI-400945 or other novel PLK4 inhibitors in TNBC. Moreover, our previous [6] and current findings support the rationale for translational and clinical studies of this combination in other breast cancer subtypes and other cancer types.