We thank Göttgens and colleagues on providing insightful comments on our article (1). We agree that vemurafenib will commonly induce G1 arrest in BRAF-mutant tumor cells, but this typically happens over the course of 12–24 hours or longer. We were observing higher levels of γH2AX within just 1 hour after radiation with 3 hours of pretreatment with vemurafenib (total = 4 hours of vemurafenib) in Fig. 3A, arguing for a direct effect of vemurafenib on altering repair of double-stranded breaks. In addition, G1-arrested cells can be radioresistant in the earlier portions of G1, compared with late G1 when they are more radiosensitive (as they approach early S-phase), so it is not clear that altered cell-cycle state is playing a direct role in radiosensitization. However, we do agree that changes in cell-cycle distribution toward a G1-arrested state could preferentially result in engagement of NHEJ DNA repair pathways, which can account for our nuclear foci repair results. Despite this, we find that expression of mutant BRAF-V600E results in increased NHEJ repair activity in our GFP reporter system in the absence of vemurafenib, but not homologous repair (HR) (Fig. 4C). In this experiment, testing the effect of BRAF-V600E and vemurafenib on NHEJ or HR activity in our GFP reporter system, we are not inducing DNA damage by radiation, but rather by the endonuclease I-SceI, which induces double-strand breaks in our reporter DNA at the recognition sequence. The reason we treated with vehicle or vemurafenib at the same time as treatment with adenoviral I-SceI was to allow for time of infection and expression of I-SceI enzyme over the course of 24 hours. In effect, this served as a period of pretreatment with vemurafenib prior to DNA damage induction by I-SceI. As shown in the right panel of Fig. 4C, we did not observe an increase in NHEJ repair activity of cells treated with 42 hours (18 + 24 hours) of vemurafenib in the empty vector (control)-transfected cells, arguing against a role for an increase in G1 fraction accounting for the differences in DNA repair. However, in cells expressing BRAF-V600E, there was a higher level of baseline NHEJ repair activity in the absence of vemurafenib, which was significantly reduced when cells were treated for 42 hours with vemurafenib.

Our findings also suggest that a mechanism for heightened DNA repair in BRAF-mutant cells may be due to alterations in XLF expression. The reason for selecting XLF as our focus for this article was that out of the NHEJ repair pathway proteins identified to be upregulated in Supplementary Table S2, we were able to verify that upregulation of XLF RNA resulted in protein upregulation by immunoblotting. We agree more in-depth study is warranted to more comprehensively assess the effects of mutant BRAF on DNA repair, particularly NHEJ repair.

See the original Letter to the Editor, p. 6556

No potential conflicts of interest were disclosed.

This work was supported by the following grants: American Cancer Society (RSG-17-221-01-TBG), NIH P50CA168505 (Thyroid SPORE CDP), and National Center for Advancing Translational Sciences (KL2TR001068 all to T.M. Williams). Research reported in this article was also supported by The Ohio State University Comprehensive Cancer Center (OSU-CCC) and NIH (P30 CA016058).

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