Dacomitinib, an irreversible pan-HER inhibitor, had shown modest clinical activity in

Dacomitinib, an irreversible pan-HER inhibitor, had shown modest clinical activity in squamous cell carcinoma of head and neck (SCCHN) patients. contexts that may have arisen due to error-prone translesional synthesis. Somatic mutations in 4.4 mutations/Mb; Wilcoxon test, and and and showed significant enrichment to dacomitinib-sensitive tumors (mutations are apparently loss-of-functional events (one missense mutation and two frameshifting indels; Fig. 4C), suggesting that the loss of functions in may confer sensitivity to dacomitinib. All three variants were not present in dbSNP135, 1000Genomes or the NIH-NHLBI6500 exome databases, indicating that these mutations may be pathogenic. We next compared the mutation frequency of and in our cohort of cisplatin-resistant SCCHN against TCGA SCCHN exomes. We observed significant enrichment of mutations in (Fisher exact test, (Fisher exact test, mutation, an alteration that is rarely observed in treatment-naive SCCHN12,19,20. Consistent with this, we observed an additional REV3L frameshift indel in one dacomitinib-sensitive tumor (T15) at a very low frequency (<5%). Strongly supporting our hypothesis, all three 151319-34-5 mutations were confirmed in cisplatin-treated samples but not in cisplatin-na?ve samples using bi-directional Sanger sequencing (Supplementary Figure S2). The final mutations were predicted at below Sangers detection level and we are unable to confirm this mutation. REV3L Inactivation Confers Sensitivity to Dacomitinib Inactivation of REV3L induces accumulation of persistent DNA damages containing unrepairable DNA double strand breaks (DSBs) in cancer cells, leading to suppression of tumor cell growth and induction of cellular senescence21. EGFR inhibitors induce cell cycle arrest and cellular senescence in tumor cells sustaining DNA DSBs by suppressing DNA repair capacity22,23. Therefore, we hypothesize that there is a favourable antitumor interaction between REV3L inactivation and dacomitinib via accumulation of unrepairable DNA DSBs. We first measured the mRNA expression of REV3L in two dacomitinib-sensitive cells (CAL27, CAL33) and two dacomitinib-resistant cells (FADU, MSKQLL2). All four cell lines showed similar levels of REV3L mRNA expressions, regardless of their sensitivity to dacomitinib, suggesting that significant REV3L depletion may be required Rabbit polyclonal to USP29 for meaningful functional effects on dacomitinib sensitivity (Supplementary Figure S3). We selected two cell lines (FADU, MSKQLL2) with strong resistance to dacomitinib (IC50 > 1?M) for further analysis. Next, we evaluated whether REV3L depletion may enhance sensitivity to dacomitinib in FADU and MSKQLL2 cells. Compared to dacomitinib or siREV3L alone, combined treatment of dacomitinib and siREV3L significantly induced G0/G1 arrest in both cells 151319-34-5 (Fig. 5A). Consistent with the effects of siREV3L on cell cycle arrest, the antiproliferative effect of dacomitinib in both cells was significantly increased by cotreatment of siREV3L in colony formation assays (Fig. 5B). Furthermore, cotreatment of dacomitinib and siREV3L significantly induced 151319-34-5 cellular senescence, as evidenced by staining of a senescence biomarker, acidic -galactosidase (Supplementary Figure S4). In contrast, synergistic antitumor effects were not observed by the combined treatment of siREV3L with paclitaxel or cisplatin, suggesting that loss of REV3L function may contribute to selective sensitization of tumor cells to dacomitinib (Supplementary Figure S5). Figure 5 Suppression of REV3L enhances sensitivity to dacomitinib in FADU and MSKQLL2 cells. Together, these data suggest that REV3L inactivation enhance response to dacomitinib by inducing cell cycle arrest and cellular senescence. Cotreatment of siREV3L and Dacomitinib produced Synergistic Antitumor effects by Inhibition of Homologous Recombination repair In addition to important roles in error-prone TLS in which DNA replication bypasses blocking lesions, REV3L has also been implicated in promoting repair of DNA DSBs by homologous recombination (HR)24,25. EGFR inhibitors have been reported to attenuate HR repair of DNA DSBs, resulting in persistent DNA damage26,27,28. Therefore, we investigated whether combination of siREV3L and dacomitinib can increase nuclear H2AX 151319-34-5 foci, which is an marker of DNA DSBs. In both cells, treatment with siREV3L alone or dacomitinib alone produced modest increase of H2AX foci. Notably, cotreatment of siREV3L and dacomitinib significantly increased H2AX foci, suggesting the significant reduction of HR-mediated DNA DSB repair (Fig. 6A). Figure 6 Dacomitinib induces persistent DNA damage in REV3L-depleted head and neck cancer cells. BRCA1 is an essential component of HR repair of DNA DSBs and the nuclear function of BRCA1 is controlled by its subcellular localization29,30. To explore inhibition of HR repair, we observed BRCA1 cytoplasmic shuttling upon treatment of siREV3L alone, dacomitinib alone or their combination. In both cells, the inhibition of REV3L alone modestly increased cytoplasmic translocation of BRCA1. Remarkably, cotreatment of siREV3L and dacomitinib resulted in significant cytoplasmic translocation of BRCA1, preventing BRCA1-induced HR repair (Fig. 6B). Cytoplasmic retention.