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. 2024 May 14:14:1380633.
doi: 10.3389/fonc.2024.1380633. eCollection 2024.

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation

Asli Muvaffak  1 , Kevin G Coleman  1
Affiliations

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation

Asli Muvaffak et al. Front Oncol. .

Abstract

Background: Ataxia telangiectasia-mutated (ATM) kinase is a central regulator of the DNA damage response (DDR) signaling pathway, and its function is critical for the maintenance of genomic stability in cells that coordinate a network of cellular processes, including DNA replication, DNA repair, and cell cycle progression. ATM is frequently mutated in human cancers, and approximately 3% of lung cancers have biallelic mutations in ATM, i.e., including 3.5% of lung adenocarcinomas (LUAD) and 1.4% of lung squamous cell carcinomas (LUSC).

Methods: We investigated the potential of targeting the DDR pathway in lung cancer as a potential therapeutic approach. In this context, we examined whether ATM loss is synthetically lethal with niraparib monotherapy. This exploration involved the use of hATM knockout (KO) isogenic cell lines containing hATM homozygous (-/-) and heterozygous (+/-) generated via CRISPR/Cas9 gene knockout technology in DLD-1, a human colorectal adenocarcinoma cell line. Subsequently, we extended our investigation to non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) models for further validation of poly ADP-ribose polymerase inhibitor (PARPi) synthetic lethality in ATM mutant NSCLC models.

Results: Here, we demonstared that biallelic hATM deletion (-/-) in DLD-1 impairs homologous recombination (HR) repair function and sensitizes cells to the PARPi, niraparib. Niraparib also caused significant tumor regression in one-third of the NSCLC PDX models harboring deleterious biallelic ATM mutations. Loss of hATM (-/-) was concomitantly associated with low BRCA1 and BRCA2 protein expression in both the hATM (-/-) DLD-1 cell line and PARPi-sensitive ATM mutant NSCLC PDX models, suggesting a downstream effect on the impairment of HR-mediated DNA checkpoint signaling. Further analysis revealed that loss of ATM led to inhibition of phosphorylation of MRN (Mre11-Rad50-NBS1) complex proteins, which are required for ATM-mediated downstream phosphorylation of p53, BRCA1, and CHK2.

Conclusions: Taken together, our findings highlight that the synthetic lethality of niraparib in ATM-deficient tumors can be regulated through a subsequent effect on the modulation of BRCA1/2 expression and its effect on HR function.

Keywords: ATM kinase; DNA double strand break (DSB) repair; PARP; genomic instability; homologous recombination deficiency; non-small cell lung carcinoma (NSCLC); synthetic lethality.

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Conflict of interest statement

Both authors, AM and KC, are employees of and hold ownership interest in GlaxoSmithKline.

Figures

Figure 1
Figure 1
DNA damage repair (DDR) pathway overview and the role of key checkpoint regulators, ATM, ATR, and DNA-PK, in DSB repair processes. Adopted from (11, 12).
Figure 2
Figure 2
Homozygous loss of ATM resulted in a marked increase in the in vitro and in vivo sensitivity of tumor cells to niraparib. Dose response curves for niraparib in BRCA2 (−/−, clone 2B4, internally generated, and Horizon Discovery, Cat. no. HD 105-007), hATM (−/−), and hATM (+/−) KO cell lines and niraparib sensitivity summary and sensitivity ranking using 2D colony formation assay (CFA), heatmap summary (A), WB analysis to confirm the level of KO in DLD-1 hBRCA2 (−/−) and hATM (−/−) (B), niraparib in vivo activity in DLD-1 hBRCA2 (−/−, Horizon Discovery), DLD-1 hATM (−/−) KO and DLD-1 hATM (+/−) KO xenografts (C), and comparison of in vitro vs. in vivo activity of niraparib in DLD-1 hBRCA2 and hATM (+/−), (−/−) KO cell lines from 3D clonogenic assay (D). In vivo activity, tumor growth inhibition% (TGI%): [1−(ΔT/ΔC)]×100; response criteria: according to NCI standards, a T/C ≤ 42% is the minimum level of anti-tumor activity and a T/C <10% is considered a high anti-tumor activity level; in vitro activity (CFA), CGI% (colony growth inhibition %): [1−(TIC50/CIC50)]×100, where TIC50 = niraparib IC50 on DLD-1 hATM KO or hBRCA2 KO and CIC50 = niraparib IC50 on DLD-1 parental.
Figure 3
Figure 3
The mean foci distribution of RAD51 (in vitro) at 3 µM of etoposide treatment in DLD-1 parental, DLD-1 hBRCA2 HOMO KO B4, and DLD-1 hATM HOMO KO at 18 h and 72 h was plotted (the mean foci count and the percentage of foci positive cells, i.e., >5 RAD51 foci/nucleus are shown. The average of the duplicate wells + SEM is shown. The percentage of Geminin-positive nuclei was plotted. Only when more than 200 Geminin-positive nuclei were identified was the percentage of RAD51 and Geminin double-positive cells plotted to minimize bias due to small sample size) (A). Changes in γH2AX and Rad51 protein levels in DLD-1 hBRCA2 (−/−) and DLD-1 hATM (−/−) KO, from niraparib treated in vivo tumors at termination (B).
Figure 4
Figure 4
Loss of ATM is associated with low BRCA and RAD51 expression and inhibition of phosphorylation of MRN complex proteins in DLD-1 hATM (−/−) KO cell line. Change in BRCA1, BRCA2, and RAD51 protein levels (by WB analysis) following vehicle and niraparib treatment (50 mg/kg PO, QD 28 days or longer) in tumors from DLD-1 hATM (−/−) KO cell line tumor model, n=6 mice/arm (A); %pRAD50(Ser638)/Rad50 and %pNBN(Ser343)/NBN expression in DLD-1 hATM (−/−) KO cell line by WB analysis, compared to levels in Dld-1 parental cell line (%pRad50(Rad50)/beta-actin), (n=1) (B), and (%pNBN(NBN)/beta-actin), (n=1) (C) (niraparib at 3 µM and etoposide at 40 µM).
Figure 5
Figure 5
Niraparib monotherapy in vivo activity summary in HRR mutant NSCLC PDX models (A), correlation analysis of tumor growth inhibition (TGI%) and downregulation of BRCA1/2 expression in the indicated ATM (−/−) PDX models; TGI%: [1−(ΔT/ΔC)]×100, following niraparib monotherapy at 50 mg/kg PO, QD for 28 days or longer, and baseline BRCA1 and 2 protein expression profiling by WB analysis; %Rad51 expression in PDX samples by WB analysis, compared to levels in Dld-1 parental cell line (%Rad51/beta-actin) (B).
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The study was funded by Tesaro/GSK, Waltham, MA. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.