Tousled-like Kinase Loss Confers PARP Inhibitor Resistance in BRCA1-mutated Cancers by Impeding Non-homologous End Joining Repair

Overview

Journal Mol Med

Publisher Biomed Central

Date 2025 Jan 23

PMID 39844055

Authors

Min-Ah Kim

Banseok Kim

Jihyeon Jeon

Jonghyun Lee

Hyeji Jang

Minjae Baek

Sang-Uk Seo

Dongkwan Shin

Anindya Dutta

Kyung Yong Lee

Affiliations

Soon will be listed here.

Abstract

Background: Double-strand breaks (DSBs) are primarily repaired through non-homologous end joining (NHEJ) and homologous recombination (HR). Given that DSBs are highly cytotoxic, PARP inhibitors (PARPi), a prominent class of anticancer drugs, are designed to target tumors with HR deficiency (HRD), such as those harboring BRCA mutations. However, many tumor cells acquire resistance to PARPi, often by restoring HR in HRD cells through the inactivation of NHEJ. Therefore, identifying novel regulators of NHEJ could provide valuable insights into the mechanisms underlying PARPi resistance.

Methods: Cellular DSBs were assessed using neutral comet assays and phospho-H2AX immunoblotting. Fluorescence-based reporter assays quantified repair via NHEJ or HR. The recruitment of proteins that promote NHEJ and HR to DSBs was analyzed using immunostaining, live-cell imaging following laser-induced microirradiation, and FokI-inducible single DSB generation. Loss-of-function experiments were performed in multiple human cancer cell lines using siRNA-mediated knockdown or CRISPR-Cas9 gene knockout. Cell viability assays were conducted to evaluate resistance to PARP inhibitors. Additionally, bioinformatic analyses of public databases were performed to investigate the association between TLK expression and BRCA1 status.

Results: We demonstrate that human tousled-like kinase (TLK) orthologs are essential for NHEJ-mediated repair of DSBs and for PARPi sensitivity in cells with BRCA1 mutation. TLK1 and TLK2 exhibit redundant roles in promoting NHEJ, and their deficiency results in a significant accumulation of DSBs. TLKs are required for the proper localization of 53BP1, a key factor in promoting the NHEJ pathway. Consequently, TLK deficiency induces PARPi resistance in triple-negative breast cancer (TNBC) and ovarian cancer (OVCA) cell lines with BRCA1 deficiency, as TLK deficiency in BRCA1-depleted cells, impairs 53BP1 recruitment to DSBs and reduces NHEJ efficiency, while restoring HR.

Conclusions: We have identified TLK proteins as novel regulators of NHEJ repair and PARPi sensitivity in BRCA1-depleted cells, suggesting that TLK repression may represent a previously unrecognized mechanism by which BRCA1 mutant cancers acquire PARPi resistance.

References

Mao Z, Bozzella M, Seluanov A, Gorbunova V . Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst). 2008; 7(10):1765-71. PMC: 2695993. DOI: 10.1016/j.dnarep.2008.06.018. View

Klimovskaia I, Young C, Stromme C, Menard P, Jasencakova Z, Mejlvang J . Tousled-like kinases phosphorylate Asf1 to promote histone supply during DNA replication. Nat Commun. 2014; 5:3394. PMC: 3977046. DOI: 10.1038/ncomms4394. View

Escribano-Diaz C, Orthwein A, Fradet-Turcotte A, Xing M, Young J, Tkac J . A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice. Mol Cell. 2013; 49(5):872-83. DOI: 10.1016/j.molcel.2013.01.001. View

Farmer H, McCabe N, Lord C, Tutt A, Johnson D, Richardson T . Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature. 2005; 434(7035):917-21. DOI: 10.1038/nature03445. View

Yamane A, Robbiani D, Resch W, Bothmer A, Nakahashi H, Oliveira T . RPA accumulation during class switch recombination represents 5'-3' DNA-end resection during the S-G2/M phase of the cell cycle. Cell Rep. 2013; 3(1):138-47. PMC: 3563767. DOI: 10.1016/j.celrep.2012.12.006. View

Krause D, Jonnalagadda J, Gatei M, Sillje H, Zhou B, Nigg E . Suppression of Tousled-like kinase activity after DNA damage or replication block requires ATM, NBS1 and Chk1. Oncogene. 2003; 22(38):5927-37. DOI: 10.1038/sj.onc.1206691. View

Kim J, Tan Y, Wang X, Cao X, Veeraraghavan J, Liang Y . Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers. Nat Commun. 2016; 7:12991. PMC: 5064015. DOI: 10.1038/ncomms12991. View

Belal H, Ng E, Meitinger F . 53BP1-mediated activation of the tumor suppressor p53. Curr Opin Cell Biol. 2024; 91:102424. DOI: 10.1016/j.ceb.2024.102424. View

Ceccaldi R, Rondinelli B, DAndrea A . Repair Pathway Choices and Consequences at the Double-Strand Break. Trends Cell Biol. 2015; 26(1):52-64. PMC: 4862604. DOI: 10.1016/j.tcb.2015.07.009. View

10.

Golding S, Morgan R, Adams B, Hawkins A, Povirk L, Valerie K . Pro-survival AKT and ERK signaling from EGFR and mutant EGFRvIII enhances DNA double-strand break repair in human glioma cells. Cancer Biol Ther. 2009; 8(8):730-8. PMC: 2863288. DOI: 10.4161/cbt.8.8.7927. View

11.

Xu Y, Xu D . Repair pathway choice for double-strand breaks. Essays Biochem. 2020; 64(5):765-777. DOI: 10.1042/EBC20200007. View

12.

Shah J, Pueschl D, Wubbenhorst B, Fan M, Pluta J, DAndrea K . Analysis of matched primary and recurrent BRCA1/2 mutation-associated tumors identifies recurrence-specific drivers. Nat Commun. 2022; 13(1):6728. PMC: 9640723. DOI: 10.1038/s41467-022-34523-y. View

13.

Chapman J, Barral P, Vannier J, Borel V, Steger M, Tomas-Loba A . RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression of DNA double-strand break resection. Mol Cell. 2013; 49(5):858-71. PMC: 3594748. DOI: 10.1016/j.molcel.2013.01.002. View

14.

Lee K, Dutta A . Chk1 promotes non-homologous end joining in G1 through direct phosphorylation of ASF1A. Cell Rep. 2021; 34(4):108680. PMC: 7941734. DOI: 10.1016/j.celrep.2020.108680. View

15.

Gunn A, Stark J . I-SceI-based assays to examine distinct repair outcomes of mammalian chromosomal double strand breaks. Methods Mol Biol. 2012; 920:379-91. DOI: 10.1007/978-1-61779-998-3_27. View

16.

Cruz C, Castroviejo-Bermejo M, Gutierrez-Enriquez S, Llop-Guevara A, Ibrahim Y, Gris-Oliver A . RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer. Ann Oncol. 2018; 29(5):1203-1210. PMC: 5961353. DOI: 10.1093/annonc/mdy099. View

17.

Toh M, Ngeow J . Homologous Recombination Deficiency: Cancer Predispositions and Treatment Implications. Oncologist. 2021; 26(9):e1526-e1537. PMC: 8417864. DOI: 10.1002/onco.13829. View

18.

Khalil M, De Benedetti A . Tousled-like kinase 1: a novel factor with multifaceted role in mCRPC progression and development of therapy resistance. Cancer Drug Resist. 2022; 5(1):93-101. PMC: 8992593. DOI: 10.20517/cdr.2021.109. View

19.

Segura-Bayona S, Knobel P, Gonzalez-Buron H, Youssef S, Pena-Blanco A, Coyaud E . Differential requirements for Tousled-like kinases 1 and 2 in mammalian development. Cell Death Differ. 2017; 24(11):1872-1885. PMC: 5635214. DOI: 10.1038/cdd.2017.108. View

20.

Lee S, Segura-Bayona S, Villamor-Paya M, Saredi G, Todd M, Stephan-Otto Attolini C . Tousled-like kinases stabilize replication forks and show synthetic lethality with checkpoint and PARP inhibitors. Sci Adv. 2018; 4(8):eaat4985. PMC: 6082654. DOI: 10.1126/sciadv.aat4985. View