Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5 0 -OH and 3 0 -phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.
Collectively, these results reveal that Elg1 forms a novel and conserved alternative RFC complex. Furthermore, we propose that genome instability arises at high frequency in elg1 mutants due to a defect in Okazaki fragment maturation.
Background: MET amplification (amp) is a resistance mechanism to EGFR TKI treatment. Emibetuzumab, a bivalent MET antibody (Ab) blocks HGF binding to MET and internalizes the receptor. Combination of emibetuzumab with EGFR TKIs (erlotinib, AZD9291, CO1686) or EGFR Ab (necitumumab, cetuximab) was evaluated in 3 ER xenograft models. Methods: Model 1: ER cell line HCC827ERL with high focal MET amp, high pMET, EGFR ex19 del (no T790M) was created from parental HCC827 NSCLC (EGFR ex19 del, EGFR amp, no MET amp) by increasing concentration of erlotinib in vitro over 7 months. Model 2: ER cell line HCC827-A8 was derived from HCC827 parental xenograft tumor serially passed in vivo with long term treatment of gefitinib and erlotinib. HCC827-A8 cells express high focal MET amp, high pMET/AXL (Western blot) while retaining EGFR ex19 del (no T790M). Model 3: LU0858 was an ER patient-derived NSCLC xenograft tumor, with focal MET amp, EGFR L858R (no T790M). MET amp and EGFRmt was determined by FISH and LNA-PCR sequencing respectively. Compound dosing: emibetuzumab 20 mg/kg qw; necitumumab 4 mg/kg or 20 mg/kg biw; cetuximab 4 mg/kg biw; erlotinib 25 mg/kg qd; 5 mg/kg AZD9291 qd; 30 mg/kg CO1686 bid. Results: EGFR inhibitors, but not emibetuzumab showed significant single agent anti-tumor effect in xenograft tumors derived from non-MET amp HCC827 parental cells. In MET amp ER models, single agent emibetuzumab resulted in tumor growth inhibition in Model 1 (T/C= 51.7%-61.0%, p<0.05) and 3 (T/C=2.8%, p<0.05)] but no tumor regression, and no anti-tumor effect in Model 2. Where evaluated, EGFR inhibitors showed no anti-tumor effect in the 3 ER models as monotherapy, except necitumumab (20 mg/kg) in Model 1 (T/C = 36.2%, p<0.05). However, combination of emibetuzumab with AZD9291, CO1686, necitumumab (20 mg/kg), or erlotinib resulted in 80.4%, 58.2%, 44.4%, 69.1% tumor regression respectively (p<0.001) in Model 1, while emibetuzumab + cetuximab (4 mg/kg) resulted in tumor stasis (T/C=0.2%, p<0.05). In Model 2, emibetuzumab + AZD9291 resulted in tumor stasis (T/C = 12.9%, p<0.05). In Model 3, emibetuzumab + necitumumab (20 mg/kg) resulted in 80.1% tumor regression (p<0.001). Conclusion: The three erlotinib resistant models with MET amp and retaining sensitizing EGFRmt (ex19 del or L858R), and no acquired T790M were found resistant to other EGFR inhibitors (Abs and TKIs). Emibetuzumab in combination with either EGFR TKI or Ab showed anti-tumor activity in MET amp ER xenograft models including tumor regression in 2 out of 3 models. The combination of emibetuzumab with erlotinib is being evaluated in NSCLC patients with EGFR activating mutation (NCT01897480). Citation Format: Suzane L. Um, Victoria L. Peek, Jennifer R. Stephens, Jessica A. Baker, Holly K. Cannon, Joel D. Cook, Isabella H. Wulur, Roger Agyei, Sudhakar Chintharlapalli, Robert J. Evans, William J. Feaver, Lysiane Huber, Linda N. Lee, Ling Liu, Liandong Ma, Ruslan Novosiadly, Volker Wacheck, Sau-Chi Betty Yan. Antitumor activity of MET antibody emibetuzumab (LY2875358) in combination with EGFR inhibitors in erlotinib resistant (ER) xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 519. doi:10.1158/1538-7445.AM2017-519
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