Reply to Oegema et al.: CFI-400945 and Polo-like kinase 4 inhibition

In “CFI-400945 is not a selective cellular PLK4 inhibitor,” Oegema et al. (1) raise thoughtful comments about our article (2). We appreciate their interest and critique. They propose that CFI-400945 activity was not from Polo-like kinase (PLK4) inhibition and argue that antineoplastic activity was through Aurora B kinase (1), rather than PLK4 inhibition (2).

Kinases often share related catalytic pockets, and targeting one kinase without affecting another is difficult to achieve. This is true for CFI-400945 (2) and centrinone, the PLK4 inhibitor (3) used by Oegema et al. (1). CFI-400945 is (by IC₅₀) 38-fold more potent in causing PLK4 inhibition than in antagonizing Aurora B kinase (4, 5). Despite their claim otherwise, this CFI-400945 property is cited (2). Indeed, CFI-400945 actions beyond PLK4 inhibition are not formally excluded (2). In addition, other findings that we discuss here question their major conclusions.

Oegema et al. (1) compare treatment with CFI-400945 to that of the PLK4 inhibitor centrinone (3). They found that CFI-400945 treatment did not confer centrosome depletion at the dosages and schedules studied (1). We do not contest that centrinone can cause centrosome depletion. However, the observed findings are likely due to bimodal PLK4 inhibition (6). Low-dose CFI-400945 treatment suppresses PLK4 autophosphorylation (2). PLK4 protein stability and activity thereby increase, leading to centriole overduplication. However, high CFI-400945 treatment dosages can fully antagonize PLK4 activity, abrogating these effects. Our findings in lung cancer cells (2) are consistent with this bimodal nature. Thus, centrosome overduplication occurred at the 20- to 50-nM concentrations, but much less so at the 500-nM dosage.

Different dosages of centrinone and CFI-400945 were used (1), making it challenging to compare these PLK4 inhibitors. Also, while 7 d of treatments are used in figure 1A of ref. 1, treatment durations are not indicated for figure 1 B and C of ref. 1. Adding to the difficulty of interpretation is that a single lung cancer cell line was examined (1). In contrast, we explored CFI-400945 effects on centrosome numbers after only 24 h of treatment (limiting off-target effects) and in diverse lung cancer cell lines (2). Likewise, lower dosages of CFI-400945 were used in our study (2) than in theirs (1). Increased centrosome numbers occurred at a dosage as low as 10 nM (2), making it improbable that Aurora B kinase inhibition after CFI-400945 treatment alone drove this response, given that its IC₅₀ is 2.8 nM for PLK4 and 98 nM for Aurora B kinase (4, 5). CFI-400945 treatments at dosages higher than 50 nM reduced the accumulation of supernumerary centrosomes (2). These findings are consistent with a bimodal inhibition of PLK4. If cytokinesis failure by Aurora B kinase inhibition were primarily responsible for CFI-400945 actions, supernumerary centrosomes should increase at higher treatment dosages, but this was not found (1, 2).

In summary, CFI-400945 can inhibit Aurora B kinase, as is cited (2). We concur with Oegema et al. (1) that PLK4 is an attractive antineoplastic target. However, antineoplastic actions of CFI-400945 (2) remain consistent with those engaged by PLK4 inhibition.