RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer

Abstract

Background BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity.Patients and methodsWe investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi.ResultsRAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor.ConclusionDetection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

A RAD 51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation

“…Mechanistically, loss of 53BP1 did not explain PARPi resistance in the three BRCA1 ‐mutated PDXs that exhibited RAD51 foci. Instead, two of them exhibited BRCA1 foci by immunofluorescence, indicative of potentially functional HRR restoration by BRCA1 hypomorphic variants (Fig C; Drost et al , ; Wang et al , ; Cruz et al , ). Altogether, these results support the use of the RAD51 assay as a predictive biomarker of PARPi response independent of the BRCA status and further demonstrate that this assay captures BRCA‐related tumors that restore HRR capacity regardless of the resistance mechanism.…”

“…First, the germline population with HRR alterations, including BRCA1/2 , PALB2 and probably other genes, such as RAD51C or RAD51D . In these patients, the RAD51 assay could be used as an enrichment biomarker to better predict sensitivity to PARPi, since restoration of the HRR pathway might have occurred and result in PARPi resistance (Konstantinopoulos et al , ; Cruz et al , ). Second, tumors with somatic alterations in HRR‐related genes, such as the PALB2 mutations described in 4% of metastatic BC (Lefebvre et al , ; Lee et al , ).…”

“…Other approaches entail the quantification of BRCA1 promoter hypermethylation, BRCA1 mRNA expression, or the detection of the HRR protein RAD51 forming nuclear foci after DNA damage, as surrogate of HRR functionality (Graeser et al , ; Naipal et al , ; ter Brugge et al , ). In these sense, we showed that, in gBRCA tumors, RAD51 foci could be detected in untreated samples and correlated with PARPi resistance regardless of the underlying mechanism restoring HRR function (Cruz et al , ).…”

Chlorambucil targets BRCA 1/2‐deficient tumours and counteracts PARP inhibitor resistance

“…Chlorambucil was selectively toxic to PDTCs lacking normal BRCA1 expression (STG201, VHIO179; http://caldaslab.cruk.cam.ac.uk/bcape/) and had a small effect on BRCA1‐proficient ones (AB521). Importantly, VHIO179, a tumour carrying BRCA1 germline truncation, is resistant to treatment with PARP inhibitors due to a MAD2L2 (REV7) inactivating mutation (Bruna et al , ; Cruz et al , ). PDTCs derived from this tumour were sensitive to chlorambucil, which supports our results obtained with the Brca1 ‐deleted mouse mammary tumour‐derived cells, upon REV7 depletion using shRNAs (Fig C).…”

RMI1 contributes to DNA repair and to the tolerance to camptothecin

“…RAD51 is an essential HR factor. The RAD51 foci reflect the accumulation of recombinase at sites of DNA damage, where it facilitates the pairing of homologous DNA sequences and strand exchange (33). To investigate the impact of RMI1 depletion on RAD51 loading after CPT treatment, we examined RAD51 focus formation with or without RMI1 depletion.…”