probes above the threshold divided by the total number of probes for autosomal chromosomes.

## Genetic instability and HRD scores

For each tumor, to evaluate genetic instability, we quantified the activity of the 17 copy number signatures described (20) with the R package CINSignatureQuantification.

For each tumor, a HRD score (HRDaCGH score), based on losses of heterozygosity (LOH), was calculated as previously described (39). A score ≥ 10 was considered as HRD-high. The percentage of genome altered was calculated as the sum of altered probes divided by the total number of probes after removing sexual chromosomes.

## Next generation sequencing

Mutation profiles were established by using targeted-NGS panel of 559 genes commonly mutated in breast cancer (40). The list of genes targeted is available in the Supplementary Information. The sequence data were aligned to the human reference genome (UCSC hg19) using Burrows-Wheeler Aligner (41). Tumor samples were sequenced at an average depth of 851× (range, 520 to 1055) for the targeted regions. Bam files were processed as described (40). Single nucleotide variants (SNVs) calling was performed with a consensus approach using 8 variants callers for the SNV (Freebayes) (42), HaplotypeCaller (43), LoFreq (44), Mutect2 (45), Pisces (46), Platypus (47), VarDict (48) and Varscan2 (49) and 10 variants callers for the indel (FreeBayes (42), HaplotypeCaller (43), LoFreq (44), Mutect2 (45), pindel (50), Pisces (46), Platypus (47), Scalpel (51), VarDict (48) and Varscan2 (49)). Variants called by less than 5 variants callers were filtered out. Then, the filtered variants were annotated with the Annotate Variation Software (ANNOVAR, version 2013-11-12). Known variants found in dbsnp129 and dbsnp137 with a variant allele frequency (VAF) superior to 1% (1000 G or ESP6500) were removed. Finally, low frequency SNVs and indels that were suspected to be false positives were systematically inspected with IGV version 2.3.32 (52, 53). Mutations were classified as "neutral" or "damaging" using the majority rule of predictor software (provided by dbnsfpm: Sift, Polyphen2, LRT, MutationTaster, MutationAssessor, FATHMM, RadialSVM, LR) as previously described (54). A "recurrent" mutation, also called "hot spot", was defined as being found more than 10 times in the Catalogue of Somatic Mutations in Cancer (COSMIC V68) database (http://cancer.sanger.ac.uk/cosmic).

## RT qPCR

Total RNA was isolated from cell lines lysed in TRIzol (Invitrogen, Fisher Scientific, Illkirch-Graffenstaden, France), while PDX tumors were lysed using Lysing Matrix D (MP Biomedicals™, Doornveld, France). Subsequently, the RNA was extracted using the RNeasy Kit (Qiagen, Les Ulis, France) following

manufacturer instructions. cDNAs were synthesized from 1µg of total RNAs using random hexamers and SuperScript III Reverse transcription (Invitrogen, Fisher Scientific, Illkirch-Graffenstaden, France). Real-time qPCR was performed on a LightCycler 480 SW 1.5 apparatus (Roche, Meylan, France) with ONEGreen® FAST QPCR PREMIX (Ozyme, Saint Cyr I'Ecole, France) and designed human specific primers (Supplementary Table 1). Results were quantified with a standard curve generated by serial dilutions of a reference cDNA preparation. GAPDH transcripts were used for normalization. The fold change in gene expression was calculated as: $Fold change = 2^{-ΔΔCT}$.

## Cell lines and CRISPR-Cas9 engineered mutants

SUM159 and SUM149 TNBC cell lines a generous gift from Dr S Ethier (MUSC, Charleston, SC), were maintained in Ham's F-12 medium (Gibco™, Fisher Scientific, Illkirch-Graffenstaden, France) supplemented with 5% FBS, 10 µg/ml insulin, 1µg/ml hydrocortisone and 1% Antibiotic-antimycotic (100X) (Gibco™, Fisher Scientific, Illkirch-Graffenstaden, France). UWB1.289PT cell line was obtained from the American Type Culture Collection (ATCC) and maintained in the 50% RPMI-1640 (Gibco™), 50% MEGM (MEGM Bullet Kit; CC-3150, Lonza, Basel, Switzerland) and supplemented with 3% FBS, 1% Antibiotic-antimycotic (100X) (Gibco™, Fisher Scientific, Illkirch-Graffenstaden, France). HCC38 cell line was obtained from ATCC and maintained in RPMI-1640 supplemented with 10% FBS and 1% Antibiotic-antimycotic.

For CRISPR-Cas9 generation of KO clones, SUM159PT cells were first transduced with a plasmid vector containing doxycycline inducible lentiviral expression of SpCas9. Lentiviral transduction was performed on 70% confluent cell cultures. Viral particles were added in the fresh medium containing 8µg/ml polybrene. After 16h the medium was changed and 2µg/ml puromycin added for cell selection for at least 5 days. Next, the cells were transduced as described above with two lentiviral plasmid vectors for the expression of sgBRCA1 (kind gift from Yea-Lih Lin, IGH, Montpellier). After lentiviral transduction, cells were selected with 400µg/ml G418 for 10 days and Cas9 expression was induced by treating a population of cells with 1µg/ml of doxycycline for 6 days. The cells were then cloned and clones verified for the KO by Sanger sequencing and western blot. All cell lines and selected clones were genetically typed by Eurofins Genomics cell line authentification (Eurofins Genomics, Les Ulis).

## Protein extraction and Western blotting

Protein extracts were prepared by lysing either tumor tissue or cell line pellets on ice for 30 min in Tris-HCl pH7.4 50mM, NaCl 100mM, NaF 50mM, β-glycerophosphate 40mM, EDTA 5mM, Triton X100 1%, Aprotinin 10mg/ml, PMSF 100mM, Leupeptin 1mM, Pepstatin 1mM, followed by a short centrifugation to pellet debris. Protein concentrations were measured using the BCA kit (Fisher Scientific, Illkirch-Graffenstaden, France) SDS-PAGE gel
electrophoresis was done on 30µg protein samples subsequently transferred onto nitrocellulose membranes (Amersham, Velizy-Villacoublay, France) and incubated overnight at 4°C with the primary antibody. Antibodies used are listed in a separate section. Membranes were then washed and incubated with the appropriate secondary antibody in 5% non-fat dry milk in PBST for 2h at room temperature and revealed by incubation with Chemiluminescent HRP Substrate (Sigma Aldrich, Saint Quentin Fallavier, France).

## Immunofluorescence

For cell lines, cells were grown on 12mm diameter slides cover slips in 24 well-plate for 24h, then drugs were added at the predetermined IC50 concentration. After 24h drugs were washed off and cells prepared as described below. For tumor tissues, 6µm cryosections were prepared from OCT embedded deep frozen tissue and mounted on Fisherbrand™ Superfrost™ Plus Microscope Slides (Fisher Scientific, Illkirch-Graffenstaden, France) and stored at -80°C until used. Cells and tumor sections were sequentially subjected to mild extraction (0.4% Triton in PBS, 5min in cold), fixation (4% PFA diluted in PBS) and blockage/permeabilization (3% BSA + 0.2% Triton in PBS, 1 hour at room temperature), incubated overnight at 4°C with the primary antibody (diluted in 3% BSA + 0.2% Triton in PBS), then with the secondary antibody (diluted in 3% BSA + 0.2% Triton in PBS, 1h at room temperature). Between each step, slides were washed 3 times with PBS. Tumor cryosections were immersed 0.1% SBB (Sigma Aldrich, Saint Quentin Fallavier, France) and 70% ethanol for 20 minutes at room temperature to reduce tissue autofluorescence and subsequently washed three times for 5 minutes PBS with 0.02% Tween 20. Stained sections or cell were counterstained with DAPI (Fisher Scientific, Illkirch-Graffenstaden, France) to stain the nuclei and cover slips were mounted with MWL4-88 (Citifluor, CliniSciences, Nanterre, France) and stored at 4°C. Antibodies used are described in the Antibody section.

Immunofluorescence images were acquired using Zeiss microscope with a 63X-immersion oil lens and generated using Zeiss Blue software. RAD51, BRCA1, 53BP1 and γH2AX nuclear foci were scored using the CellProfiler image analysis software (version 2.2.0, Broad Institute). At least three biological replicates of each model (both vehicle- and olaparib-treated) were analyzed. Cells presenting >5 foci/nucleus for RAD51, BRCA1, 53BP1 or γH2AX were considered positive and tumors presenting >10% of positive cells were scored positive for the given marker.

## Antibodies

Immunofluorescence; rabbit anti-RAD51 PC130 1:300 (Merck Millipore Sigma Aldrich, Saint Quentin Fallavier, France), rabbit anti-geminin 52508 1:200 (CST OZYME, Saint Cyr l'Ecole, France), mouse anti-BRCA1 sc-6954 1:100 (SCBT, Heidelberg, Germany), mouse anti-γ-H2AX (H2-3F4, kind gift from Dr. Mustapha Oulad-Abdelghani, MAB-IGBMC Illkirch-Graffenstaden, 1:4000), rabbit anti-53BP1 NB100-304 1:500 (Bio-technique LTD, Abington, UK).

Secondary antibodies; goat anti-mouse Alexa Fluor 488 (Abcam ab150113, 1:1000), goat anti-rabbit Alexa Fluor 555 ab150078 1:1000 (Abcam, Cambridge, UK).

Western blotting; BRCA1 9010 1:500 (CST OZYME, Saint Cyr l'Ecole, France), BRCA2 A303-434A 1:1000 (Bethyl OZYME, Saint Cyr l'Ecole, France), PARP1 WH0000142M1 1:1000 (Sigma Aldrich, Saint Quentin Fallavier, France), RAD51 8875, 1:1000 (CST OZYME, Saint Cyr l'Ecole, France) and alfa tubulin T9026 1:20000 (Sigma Aldrich, Saint Quentin Fallavier, France); secondary antibodies goat anti-mouse-HRP 70745 1:10000 (CST OZYME, Saint Cyr l'Ecole, France) and goat anti-rabbit-HRP 7076 1:10000 (CST OZYME, Saint Cyr l'Ecole, France).

## Results

### PDX models show variable response to olaparib

To test the relative sensitivity of TNBC with silenced BRCA1 due to promoter hypermethylation (designated hereafter BRCA1-Me) to the PARP inhibitor olaparib and assess the impact of neoadjuvant treatment (NACT) on olaparib sensitivity, we selected 8 PDX models (7 TNBC and 1 High Grade Serous Ovarian Carcinoma) showing different BRCA1 profiles. Our experimental PDX set comprised 2 BRCA1 wild type (BRCA1-WT) TNBC (b1995, b3804), 4 BRCA1-Me TNBC (b3977, b4122, 15b1516, 15b0018) and 2 (1 TNBC and 1 HGSOC) BRCA1 mutated (BRCA1-Mut) models (tm168, o10047) (Table 1; Supplementary Figure 1). BRCA1-Me PDX models 15b1516, 15b0018 and b3977 were established from post-NACT residual tumors that had shown poor response to neoadjuvant treatment (Table 1; Supplementary Figure 1). The two BRCA1-Mut models (tm168, o10047) and the remaining BRCA1-Me model (b4122) were established from treatment naïve tumors. Hypermethylation of the BRCA1 promoter region was determined in both the PDX models and the patient tumors using methylation specific PCR (MS-PCR). It is of note that no pre-treatment biopsy was available for the tumors that had undergone NACT, hence, BRCA1 promoter methylation was determined after NACT on these tumor samples. Of the four BRCA1-Me PDX, 3 presented homozygous (Me/Me) methylation (15b0018, b3977, b4122) and 1 hemizygous (Me/U) methylation (15b1516) (Supplementary Figure 2).

PDX were grafted subcutaneously on Swiss-nude mice and olaparib was administered orally at 100 mg/kg, 5days per week for 5 weeks. In a parallel treatment group, mice received 50mg/kg Carboplatin (CBP) by intraperitoneal injection twice a week for 4 weeks, while the control group received daily the olaparib vehicle administered orally. Tumor volumes were measured twice a week. Three (tm168, b4122, o10047) of the 8 PDX showed disease stabilization or limited tumor size reduction under olaparib treatment, while the 5 other models (b3804, 15b0018, 15b1516, b1995, b3977) progressed (Figures 1A, B; Supplementary Figure 3A). Of the 3 olaparib responders, PDX tm168 and o10047 were BRCA1-Mut and b4122 was BRCA1-Me, but established from a treatment naïve tumor. The 3 other BRCA1-Me models (15b0018,
<figure><img src="image_146.png" /><figcaption>FIGURE 1
Tumor volume change of the 8 PDX models under olaparib and Carboplatin treatment and principal characteristics related to HR of the PDX models (A) Waterfall plot of tumor volume changes in the 3 experimental arms were computed at the end of treatment for individual tumors as the percentage of the starting volume (green vehicle, pink olaparib, grey Carboplatin) (B) mean tumor volume change in each PDX model treated with olaparib. (C) mean tumor volume change in each PDX model treated with Carboplatin. Blue and orange lines indicate the +20% and -30% thresholds for tumor response defined by the RECIST criteria; > 20% progressive (PD), < 20% - > 30% stable disease (SD), < 30% responsive (R).</figcaption></figure>

15b1516 and b3977, which progressed under olaparib were established from tumors that had received NACT. In the CBP arms we globally noted more favorable response patterns, with 6 of 8 PDX models showing partial to complete response and 2 PDX (b3804, 15b1516) progressing under treatment (Figures 1A, C; Supplementary Figure 3A). Tumor growth was monitored after treatment end in 3 PDX, 2 *BRCA1-Me* TNBC (b3977, b4122) and 1 *BRCA1-Mut* HGSOC (o10047) (Supplementary Figure 3B) Both *BRCA1-Me* TNBC resumed growth shortly after end of olaparib administration. By contrast, the *BRCA1-Mut* o10047 exhibited complete regression 25 days after treatment ended and recurred after a lull of 3 weeks. In the CBP arm, progression after treatment end was observed in 1 of 3 PDX (b4122), while the two other models did not recur during the follow up period (Supplementary Figure 3B).

## Genomic profiles and genetic instability scores of the tested TNBC models

Targeted sequencing was performed on the 7 TNBC PDX to search for mutations affecting principal DNA damage response genes and targetable cancer genes. The HGSOC PDX was analyzed by exome sequencing as part of a previous study (55). Mutations were

found in the TP53, PTEN, KRAS, RIF1 and STK11 genes (7, 2, 1, 1 and 1 PDX respectively) (Supplementary Table 2). No mutations were detected in further DNA repair genes such as BRCA2, PALB2 or the RAD51 orthologs RAD51B, C or D. The 8 PDX models were also analyzed for copy number changes by array-CGH. Genetic instability and HRD scores were determined (Figure 2A). All TNBC models, irrespective of BRCA1 mutation or epigenetic silencing, presented elevated genetic instability scores (CX2, CX5) shown to be linked with BRCA-deficiency (20). We also noted that all tested TNBC models presented elevated HRD scores as defined by Abkevich and coworkers (39). Thus, elevated genetic instability and HRD scores suggested preexisting HR deficiency in the PDX models used in this study, including the two BRCA1-WT models.

We took advantage of the CGH analysis to determine copy number changes affecting key repair genes in the TNBC models. Except b3804, most PDX presented hemizygous losses affecting 3 to 4 HR genes of a list including BRCA1, BRCA2, PALB2, RAD51, RAD51B and RAD51C suggesting that the combined copy number reductions on these key HR genes could have contributed to a global HR attenuation and elevated genetic instability in these tumors (Supplementary Figure 4). But we cannot exclude the existence of undetected genetic or epigenetic anomalies affecting genes involved in HR maintenance.
<figure><img src="image_147.png" /><figcaption>FIGURE 2
Principal characteristics related to HR of the PDX models. (A) CX Chromosomal instability and HRD scores of each PDX model. Color shades indicate from white to red the level of instability. (B) Protein expression patterns were assessed by Western blotting. The BRCA1 status of each model is indicated on top of the autoradiograms. WT wild type. Me/Me homozygous methylation. U/Me hemizygous methylation. Mut coding mutation. For each PDX model three independent extracts from individual PDX tumors were loaded and analyzed. Proteins analyzed are indicated on the left and Molecular Weights in KDa on the right. The protein extract of SUM159 cell line extract was added to the BRCA1 panel to illustrate the full length and the Δ11 BRCA1 protein variants.</figcaption></figure>

Principal genes of the BRCA pathway
showed protein expression profiles
concordant with olaparib response

To determine the impact of promoter hypermethylation or
coding mutations on BRCA1 protein expression, BRCA1 protein
levels were measured by western blotting (WB) in the 8 PDX
models. Noticeably, BRCA1-Me PDX b4122, b3977, 15b0018 and
BRCA1-Mut o10047, tm168 showed no detectable BRCA1 band,
suggesting a loss of BRCA1 functionality in these tumors. By
contrast, the BRCA1-WT b1995, b3804 and the BRCA1-
hemimethylated 15b1516 PDX presented detectable BRCA1
bands (Figure 2B). We noted that PDX b3804 expressed high
levels of a short 100 kD BRCA1 protein isoform and low levels of
the full length (268 kD) protein (Figure 2B). The 100 kD band is
compatible with the size of the hypomorphic Δ11-BRCA1 isoform,
which corresponds to a BRCA1 splice-variant where exon 11 is
excluded and that is frequently expressed in tumors bearing
mutations in exon 11 (56). However, no BRCA1 coding mutation,
that could have explained the expression of the Δ11-BRCA1
isoform, was detected in PDX b3804 (Supplementary Table 1).
We also analyzed expression of the BRCA2, RAD51, PARP1 and
53BP1 proteins, which are important actors of HR. Whereas
RAD51 expression appeared elevated and relatively constant in
the different PDX models, that of BRCA2 was more variable, with
no obvious link with BRCA1 status or olaparib response, except

possibly the *BRCA1-Mut* PDX tm168, which showed very low
BRCA2 protein levels (Figure 2B). PARP1 protein expression was
detected in all PDX models, but, interestingly, the lowest levels were
detected in the 3 *BRCA1-Me* (b3977, 150018 and 15b1516) that had
been exposed to NACT and responded poorly to olaparib
(Figure 1A). Finally, 53BP1 protein was expressed at high levels
in 5 models and at low levels in 3 other PDX (Figure 2B).

BRCA1, RAD51 and 53BP1 nuclear foci
formation in treated tumors correlate with
olaparib response

In HR-proficient cells, BRCA1 and RAD51 proteins cluster at
DNA damage sites forming nuclear foci that can be detected by
immunofluorescence labeling. Absence of BRCA1 and/or RAD51
foci is considered as a sign of HR-deficiency. We scored the fraction
of cells positive for BRCA1 and RAD51 foci in cryosections of PDX
models sampled at the end of olaparib and vehicle treatment and
searched for an association with olaparib response. Tumors
presenting >10% cells showing >5 BRCA1 or RAD51 per nucleus
were scored positive. PDX sections were immunolabeled with
commercial antibodies directed against BRCA1, RAD51, and the
S phase marker Geminin. Because of secondary antibodies species
compatibility, BRCA1 and RAD51 were co-immunolabeled
(Figure 3A; Supplementary Figure 5), whereas Geminin staining
was performed separately to ascertain the presence of S phase cells in each sample. Immunolabeling scoring was performed in 3 independent PDX tumors per model on at least 100 cells per section. Geminin staining showed 10 to 30% of Geminin-positive cells, confirming that tissue section actually comprised S phase tumor cells (Supplementary Figure 6). Four (4) of the 8 olaparib treated PDX, corresponding to 2 *BRCA1-Mut* (tm168, o10047) and 2 *BRCA1-Me* (b3977, b4122), scored negative for BRCA1-foci (Figures 3B, J), in agreement with the absence of BRCA1 band in the WB analysis (Figure 2B). BRCA1-foci positive models comprised two *BRCA1-WT* (b3804, b1995) and two *BRCA1-Me* PDX (b15b1516, 15b0018). Three of the four BRCA1-foci negative PDX (tm168, o10047, b4122) scored negative for RAD51-foci (Figures 3A, C, J, K) and corresponded to the 3 models that responded to olaparib (Figures 1A, B). Noticeably, PDX b3977, which progressed under olaparib (Figures 1A, B), scored positive for RAD51-foci, despite the fact it did not show BRCA1-foci (Figures 3A, C, J, K). All other RAD51-foci-positive PDX scored also positive for BRCA1 foci and showed mediocre response to olaparib (Figures 1A, B; 3A–C, J, K). Noticeably, the 3 *BRCA1-Me* models established from NACT treated TNBC (15b0018, 15b1516, b3977) were RAD51-foci positive and showed scores similar to those observed in the 2 *BRCA1-WT* models (b1995, b3804) (Figures 3B, J). By contrast, the *BRCA1-Me* PDX b4122, which was not exposed to NACT prior PDX establishment, was both BRCA1 and RAD51-foci-negative and did not progress under olaparib. These data suggested that while b4122 was indeed HR deficient, the 3 NACT treated *BRCA1-Me* models were, at least partially, HR proficient.

We verified whether BRCA1 or RAD51 nuclear foci formation was associated with olaparib response (Figures 3F-1). We noted a significantly association of BRCA1 and RAD51 foci absence (or low levels) with olaparib response (t-test p=0.0017 and p<0.0001 respectively) (Figures 3F, G). Interestingly, the differences in foci positive cells between responsive and non-responsive cells were discernible in both tumor sections from olaparib treated (Figures 3F, G) and sections from vehicle treated tumors and showed equivalent statistical significance (Figures 3J, K).

We also scored γH2AX and 53BP1 foci, which are standard DNA damage markers, and observed that models presented 20 to 60% foci-positive cells for either marker, indicating severe DNA damage in olaparib treated PDX, to the exception of 15b1516 where only 5 to 10% cells scored positive (Figures 3D, E). Interestingly, we noted that 53BP1-foci tended to be more frequent in olaparib responsive PDX, compared with non-responsive PDX (t-test p=0.028) (Figure 3H). However, no difference was found with γH2AX-foci (Figure 3I).

## Acquired olaparib resistance in BRCA1 deficient cell lines is associated with increased BRCA1 and RAD51 foci formation and reduced levels of DNA damage

It is well documented that BRCA-deficient tumors or cell lines rapidly acquire resistance to treatment associated with the

restoration of HRR (56). We were, thus, interested to explore the restoration of RAD51-foci formation in *BRCA1*-deficient cell lines with acquired olaparib resistance. We, thus, isolated olaparib resistant clones from three cell lines; SUM159-KO1 and SUM159-KO2, two CRISPR *BRCA1* knock out clones we engineered from the TNBC SUM159 cell line, the SUM149 TNBC cell line which bears a frameshift mutation in exon 11 (2288delT) (57) and the UWB1.289 ovarian cancer cell line also showing a frameshift mutation in exon 11 (2594delC) (58). Resistance was obtained by exposing cell cultures to incremental olaparib concentrations for at least 12 weeks. Olaparib resistant cell lines were designated with the suffix Re (SUM159-KO1-Re, SUM159-KO2-Re, SUM149-Re and UWB1.289-Re). We characterized BRCA1, BRCA2, RAD51 and PARP1 protein expression by WB, as well as RNA expression changes associated with the acquisition of olaparib resistance (Figures 4A, B). In SUM159-KO1 and SUM159-KO2 no BRCA1 protein was detected. SUM159-KO1-Re reexpressed the full length BRCA1 protein and SUM159-KO2-Re showed no difference in BRCA1 protein expression compared with it clone of origin. SUM149 and UWB1.289 expressed no full length BRCA1 and variable levels of the Δ11-BRCA1 100 kD band. Interestingly, SUM149-Re expressed the BRCA1 full length and increased levels of the Δ11-BRCA1 variant, whereas UWB1.289-Re showed strongly increased Δ11-BRCA1 levels (Figure 4A). No significant difference was found for BRCA2, RAD51 and PARP1 protein expression in the different cell line models. At the RNA expression level, we noted increased gene expression of *RAD51* and *RAD51C* in SUM159-KO1, of *RAD51C* and ABCG2 in SUM149-Re, as well as of Δ11-BRCA1, RAD51C, RAD51D, PALB2 and ATM in UWB1.289-Re which could be related with their acquisition of olaparib resistance.

Next, we scored BRCA1, RAD51, 53BP1 and γH2AX foci formation in the olaparib treated cell lines (Figure 5A). Except in SUM159-KO2-Re which does not express BRCA1, nor Δ11-BRCA1, and scored BRCA1-foci-negative, BRCA1-foci scores tended to increase in olaparib resistant clones compared with cells or origin (Figure 5B). A similar trend was noticeable for RAD51-foci, whose numbers nearly doubled in olaparib resistant cells relative to cell lines of origin (Figure 5C). By contrast, 53BP1 and γH2AX foci numbers clearly decreased in olaparib resistant variants (Figures 5D, E). These differences in foci numbers between olaparib resistant cells and their original counterparts, increase for BRCA1 and RAD51, decrease for 53BP1 and γH2AX, were all statistically significant and in coherence with findings we made on PDX (Figures 5F-1). The reduction of γH2AX-foci associated with the increase of BRCA1 and RAD51-foci observed in resistant cells suggest a global decrease of DNA damage in these cells upon olaparib treatment due to restored HR capacity. We also derived olaparib resistant cells from the hemimethylated *BRCA1-Me/UM HCC38* TNBC cell line, whose methylation status we confirmed. While BRCA1-foci scores only modestly increased in olaparib treated cells, the fraction of RAD51-foci positive cells doubled in HCC38-Re variant cells (Supplementary Figure 7). Overall, our cell line data strongly support the notion that resistance to treatment and particularly to olaparib in BRCA1-deficient cancer cells is frequently associated with restoration of RAD51 foci formation, thus, signing for restored HR capacity.
<figure><img src="image_148.png" /></figure>

<figure><img src="image_149.png" /></figure>

<figure><img src="image_150.png" /></figure>

<figure><img src="image_151.png" /></figure>

<figure><img src="image_152.png" /></figure>

<figure><img src="image_153.png" /></figure>

<figure><img src="image_154.png" /></figure>

<figure><img src="image_155.png" /></figure>

<figure><img src="image_156.png" /></figure>

<figure><img src="image_157.png" /></figure>

<figure><img src="image_158.png" /><figcaption>FIGURE 3</figcaption></figure>

BRCA1, RAD51, γH2AX and 53BP1 nuclear foci formation in olaparib treated PDX. (A) Representative immunofluorescence images of frozen PDX tissue section harvested from mice sacrificed after the last administration of olaparib or before the tumor reached ethical size for vehicle treated models. PDX tumor sections were ordered from worst to best olaparib response. A complete version including vehicle treated tumors is visible in Supplementary Figure 5. (B) Quantification of BRCA1, (C) RAD51, (D) γH2AX, (E): 53BP1 foci formation in olaparib treated PDX. Results are represented as % of foci positive cells in the analyzed tumor sections in each PDX. Cells presenting at least 5 foci/nucleus were considered positive. At least 100 cells were quantified in each tissue section. (F-I): by two-tailed unpaired t-test analyses of correlation between foci numbers and olaparib response. Nuclear foci analyzed are indicated on top of each graph, as well as p-values. * * * * * * * * * on top of the graph indicate the level of significance of the t-test (J, K) Histograms showing the percentage of BRCA1- (J) and RAD51-(K) foci positive cells of BRCA1 and RAD51 nuclear foci quantification in vehicle and olaparib treated PDX.
<figure><img src="image_159.png" /><figcaption>FIGURE 4</figcaption></figure>

Protein and mRNA expression profiles of principal HR genes in the cell line models (A) Western blots showing protein expression patterns; analyzed proteins are indicated on the left and molecular weights in kDa on the right. (B) mRNA expression changes of genes potentially associated with olaparib resistance in cell line models selected for acquired olaparib resistance. mRNA levels are expressed as fold changes of the expression levels measured between the olaparib-resistant cell line and their parental lines used as reference (horizontal line). Reference cell lines were SUM159-KO1, SUM159-KO2 parental SUM149 and parental UWB1.289.

## BRCA1, RAD51, γH2AX and 53BP1 nuclear-foci as predictors of the response to olaparib or carboplatin

As PDX responding to CBP represented twice the number of olaparib responders (6 vs. 3 respectively), we wanted to determine whether the response to CBP was associated with reduced RAD51 and/or BRCA1-foci formation (Figures 1B, C). Like in the olaparib arm, individual grafts that responded to CBP were predominantly BRCA1-foci-negative/RAD51-foci-negative (Figures 6A, B) and, interestingly, most grafts of the BRCA-foci-negative/RAD51-foci-positive model b3977, whose response to olaparib was mediocre, showed tumor size reduction under CBP. However, grafts of the BRCA1-positive/RAD51-positive PDX 15b0018 and b1995, respectively BRCA1-WT and BRCA1-Me and bad responders to olaparib, showed good response to CBP pointing to the fact that CBP efficacy is not solely based on the HR status, but can also rely on alternative DNA repair mechanisms (Figure 6B). Despite these two BRCA1-foci-positive/RAD51-foci-positive models, both BRCA1-foci-negativity and RAD51-foci-negativity were significantly associated (t-test p-value 0.0059 and 0.0176 respectively) with CBP response in our dataset (Figure 6D). Next, we computed the Sensitivity, Specificity and Accuracy of RAD51, BRCA1, 53BP1 and γH2AX foci in predicting the response to olaparib and CBP (Table 2). Concerning olaparib response RAD51 foci showed high sensitivity (88%), specificity (82%) and accuracy (84%), while BRCA1 foci showed excellent sensitivity (94%) but lower specificity (66%) and accuracy (74%). Interestingly, when CBP response was considered, BRCA1 foci performed globally better showing 70% sensitivity, 92% Specificity and 75% accuracy in comparison with RAD51 foci, which showed 57% sensitivity, 100% Specificity but 68% accuracy. The performances of 53BP1 and γH2AX foci were overall rather contrasted showing excellent sensitivity (100%), but poor specificity (18%) and accuracy (41%) for olaparib response, while they reached better values for CBP response with high accuracy (87%) mitigating the mediocre specificity (46%). Overall,

these data support the use of RAD51 foci for olaparib response prediction, while BRCA1 foci, possibly in combination with RAD51 foci, appear interesting for CBP response. While γH2AX and 53BP1 foci do not appear as convincing it may be interesting to reevaluate their performance for CBP response on a larger dataset.

## Discussion

About one TNBC in three is estimated to be HR deficient. HRD has been linked with genetic or epigenetic impairment of genes belonging to the BRCA-pathway such as BRCA1, BRCA2, PALB2 and RAD51B, RAD51C and RAD51D, but BRCA1 is the most frequently affected gene in TNBC (5, 6). Because HR deficient tumors show increased sensitivity to PARPi or Platinum salts, detection of HRD has important implications in treatment definition (59). Identification of HRD is generally based on targeted sequencing of commonly mutated HR genes, combined with the determination of patterns of genomic rearrangements typical of HRD, such as the genomic HRD score, Tandem Duplication score or CX scores (20, 27, 31). However, BRCA-deficient cancers frequently develop resistance to treatment associated with HR restoration. Involved molecular events range from reverting secondary mutations, gene rearrangements producing gene chimeras, loss or mutations of the 53BP1 gene or of one of its cofactors in the Shieldin complex (34, 35, 59). Hence, the actual status of residual HRD tumors that have previously been exposed to genotoxic treatment could be in question. This point has been specifically raised concerning TNBC with silenced BRCA1 gene due to hypermethylation of the promoter, whose sensitivity to PARPi has been disputed (31, 34).

These questions motivated the present study, where we interrogated the sensitivity to olaparib and CBP of 4 TNBC PDX models with epigenetically silenced BRCA1, 3 of which had received NACT prior PDX establishment. Noticeably, despite obliterated or severely reduced BRCA1 protein expression levels, the 3 BRCA1-
<figure><img src="image_160.png" /><figcaption>FIGURE 5
BRCA1, RAD51, γH2AX and S3BP1 nuclear foci in BRCA1 deficient cell line models (A) Representative immunofluorescence images of cell line models treated or not (control) with Olaparib for 24h. Cell line names are indicated on top BRCA1-WT (WT), BRCA1-KO (KO), BRCA1-KO-Ola-Resistant (Re), SUM149, parental (PT), Ola-Resistant (Re), UWB1289, parental (PT), Ola-Resistant (Re) (B) BRCA1, (C) RAD51, (D) S3BP1, (E) γH2AX foci formation in the respective olaparib treated cell lines. Results are represented as % of foci positive cells in the analyzed tumor sections in each PDX. Cells presenting at least 5 foci/nucleus were considered positive. At least 150 cells were quantified in each section. Representative IF images are shown in the Supplementary Figures. (F-I): Box plot presenting the difference in the number of BRCA1- (F), RAD51- (G), S3BP1- (H) and γH2AX- (I) foci positive cells between olaparib resistant and sensitive cell lines. p-values were calculated with two-tailed unpaired t-test. * , ** , *** on top of the graph indicate the level of significance of the t-test</figcaption></figure>

methylated PDX that had undergone NACT responded poorly to
olaparib, showing response profiles similar to those of the two
BRCA1-WT models used as controls. These observations, thus,
questioned the actual functionality of HR in the BRCA1-Me PDX
models included in our study, leading us to test for BRCA1 and
RAD51 nuclear foci formation in tissue sections of olaparib treated
PDX and use this assay as a functional read out. In HR proficient
cells, the BRCA1 and RAD51 proteins cluster onto DNA lesions and

these clusters can be detected as nuclear foci by
immunofluorescence microscopy (60). Their presence in tumor
tissues signs for HR proficiency (61). Remarkably, the three
BRCA1-Me PDX that responded poorly to olaparib scored
positive for BRCA1 and/or RAD51 foci, likewise the two BRCA1-
WT models. By contrast, the three PDX models responding to
olaparib (2 BRCA1-Mut and 1 BRCA1-Me) scored negative for
BRCA1 and RAD51-foci. Noticeably, the BRCA1 and RAD51 foci
<figure><img src="image_161.png" /></figure>

<figure><img src="image_162.png" /></figure>

<figure><img src="image_163.png" /></figure>

<figure><img src="image_164.png" /><figcaption>FIGURE 6</figcaption></figure>

BRCA1 and RAD51 scores are good predictors of olaparib and Carboplatin response. Waterfall plots of the tumor volume change in individual PDX models (percentage of the starting volume) treated with either olaparib or Carboplatin. The status according to BRCA1 and/or RAD51 foci formation of each model is indicated by the color of the bar; dark blue BRCA1+/RAD51+; light blue BRCA1-/RAD51+; red BRCA1-/RAD51-. The identity of the PDX model is indicated by color boxes at the bottom of the graph. (A) olaparib treated mice. (B) Carboplatin treated mice. (C) tumor volume change under olaparib treatment stratified according to the BRCA1 foci status. (D) tumor volume change under olaparib treatment stratified according to the RAD51 foci status. *, **,*** on top of the graph indicate the level of significance of the t-test.

negative BRCA1-Me model had never been exposed to chemotherapy. Thus, our data suggest that HR was, at least partially, functional in the BRCA1-Me PDXs established from residual TNBC previously exposed to NACT, contributing to their poor response to olaparib. These observations were consistent with the increased RAD51 foci formation and HR restoration we evidenced in BRCA1-deficient cell line models that we rendered olaparib-resistant. Results presented herein support the notion that the actual HRD status of BRCA1-Me TNBC may be in doubt, especially if they have been previously exposed to genotoxic treatment. Since we did not have access to tumor samples

prior NACT, we cannot conclude on HR restoration due to treatment exposure, but our data are in line with previous reports (31, 35). This calls for the verification of the HRD status based on functional read outs such as BRCA1 and/or RAD51 foci formation (62). Indeed, genetic tests or genomic scores yield valuable information on the HRD status of a given tumor, however, they point to its natural history and may be misleading in terms of actual HR functionality. We wish, moreover, to point out that all the PDX models included in our study presented elevated HRD or genetic instability scores, irrespective of their mutational, BRCA1 methylation status or sensitivity to olaparib.

<table><caption>TABLE 2 Test performance values for the indicated HRD biomarkers to predict olaparib and CBP response.</caption><thead><tr><th></th><th>Biomarkers</th><th>RAD51 Foci (n=61)</th><th>BRCA1 Foci (n=61)</th><th>γH2AX Foci (n=61)</th><th>53BP1 Foci (n=61)</th></tr></thead><tbody><tr><td rowspan="5">Olaparib response</td><td>Sensitivity</td><td>88%</td><td>94%</td><td>100%</td><td>100%</td></tr><tr><td>Specificity</td><td>82%</td><td>66%</td><td>18%</td><td>18%</td></tr><tr><td>PPV</td><td>65%</td><td>52%</td><td>32%</td><td>32%</td></tr><tr><td>NPV</td><td>95%</td><td>97%</td><td>100%</td><td>100%</td></tr><tr><td>Accuracy</td><td>84% (n=53)</td><td>74% (n=53)</td><td>41% (n=53)</td><td>41% (n=53)</td></tr><tr><td rowspan="5">CBP response</td><td>Sensitivity</td><td>57%</td><td>70%</td><td>100%</td><td>100%</td></tr><tr><td>Specificity</td><td>100%</td><td>92%</td><td>46%</td><td>46%</td></tr><tr><td>PPV</td><td>100%</td><td>97%</td><td>85%</td><td>85%</td></tr><tr><td>NPV</td><td>43%</td><td>50%</td><td>100%</td><td>100%</td></tr><tr><td>Accuracy</td><td>68%</td><td>75%</td><td>87%</td><td>87%</td></tr></tbody></table>

Response includes disease stabilization.
Genetic and genomic scores are being considered concurrently with RAD51 foci determination as biomarkers predictive of treatment in TNBC and prostate cancer (63–65). Results support the excellent correlation between low RAD51 scores and HR deficiency, as well as with increased sensitivity to olaparib or platinum based regimen (63, 65, 66). However, these works also point out the detection of tumors showing poor response to treatment, whilst presenting inactivating BRCA1 or BRCA2 mutations or elevated HRD scores and scoring positive for RAD51 foci. Our data are in perfect concordance with these observations and further support the relevance of BRCA1 and/or RAD51 foci-based tests to determine the functionality of HR in TNBC presenting all signs or HRD (63, 65, 66). Further, our data highlight the importance of verifying the actual functionality of HRR in tumors with epigenetically silenced BRCA1, particularly those previously exposed to chemotherapy during the course of the disease.

A number of studies highlight the exquisite sensitivity of HRD
tumors to cis or carboplatin, alone or in combination with other
molecules (63, 65, 66). We thus, documented the sensitivity to CBP
of our models and determine their overlap with BRCA1 and RAD51
scores. PDX models responding to CBP were twice more frequent
than olaparib responders and included all BRCA1-foci negative
cases, as well as 2 BRCA1-foci/RAD51-foci positive PDX models,
underlining the fact that CBP sensitivity is not solely determined by
the HRD status. Indeed, platinum salts produces bulky adducts that
must be removed by NER to avoid DNA breaks and tumors with
faulty NER have been shown to be highly sensitive to platinum (59,
67, 68). Hence, while BRCA1 foci appeared a better predictor of
CBP sensitivity than RAD51 in our dataset, they missed 2 out 6 CBP
sensitive cases, thus calling for complementary tests.

Conclusion

Our work shows that TNBC with a silenced *BRCA1* gene, due to hypermethylation of its promoter, may be prone to HR restoration and, thus, become resistant to olaparib. Interestingly, two of the olaparib resistant and RAD51-foci positive PDX models appeared sensitive to CBP. Our data thus support the notion that the HRD status of TNBC should be systematically checked using a combination of biomarkers, among which the BRCA1 and RAD51 foci formation tests play a major role. Not only do these assays inform on HR functionality in a given tumor, they are cheap, rapid and quite easy to implement. We performed the immunofluorescence analysis on olaparib treated tumor samples to ensure a clear signal difference between foci positive and foci negative samples, but noted, in accordance with other works, that the difference was also perceptible in non-treated tumors (63). Hence, this test, which we and others have shown to reliably predict sensitivity to olaparib, and also to CBP, could be implemented on Formalin fixed tumors as part of a pathology routine in a number of cancer treating institutions.

Data availability statement

The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.

Ethics statement

The animal study was reviewed and approved by CEEA-LR-12028.

Author contributions

Conceptualization: CT. Funding acquisition: WJ and CT. Investigation: CV, EO, IT, LF, JA, and EC. Methodology and resources: BO, MC, and AG. Supervision: CT and WJ. Validation: DB, CS, WJ, and CT. Writing: CT and WJ. All authors contributed to the article and approved the submitted version.

Funding

The authors declare that this study received funding from Astra-zeneca (contract # 2018-02069). 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. This work also benefited from the following financial support: Institut National du Cancer PRTK-2017 MODUREPOIS, Ligue Nationale Contre le Cancer 'Comité régional Occitanie-Est' 2021-R22031FF and the SIRIC Montpellier Cancer Grant INCa-DGOS-Inserm_12553.

Acknowledgments

The authors sincerely acknowledge Dr Genevieve Rodier for
fruitful discussions and help throughout this project, Dr Isabelle
Jariel for critical reading and constructive comments on the
manuscript, and Dr Rui-Bras Gonçalvez and Dr Stanislas du
Manoir for help during the establishment of the PDX models.
Furthermore, sincere thanks are due to the staffs of the animal
facility at IRCM and the Centre de Ressources Biologiques at ICM
for their constant support and expert help.

Conflict of interest

The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fonc.2023.1125021/
full#supplementary-material
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Annexe 2.

# Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy?
Review

# Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy?

Esin Orhan <fnref n="1" />, Carolina Velazquez <fnref n="2" />, Imene Tabet <fnref n="1" />, Claude Sardet <fnref n="1" /> and Charles Theillet <fnref n="1" />,<fnref n="2" />,*

**Simple Summary:** RAD51 is an essential gene for cell survival. Its function is central to DNA repair and it protects cells from life-threatening damage to the genome. Interestingly, RAD51 is expressed at high levels in a large proportion of cancers, and elevated RAD51 expression is associated with a bad outcome and reduced response to treatment. Hence, reducing RAD51 expression and/or interfering with its function could be of great therapeutic value. We review here the multiple levels of regulation of RAD51 expression and function and explore potential therapeutic leads.

**Abstract:** The RAD51 recombinase is a critical effector of Homologous Recombination (HR), which is an essential DNA repair mechanism for double-strand breaks. The RAD51 protein is recruited onto the DNA break by BRCA2 and forms homopolymeric filaments that invade the homologous chromatid and use it as a template for repair. RAD51 filaments are detectable by immunofluorescence as distinct foci in the cell nucleus, and their presence is a read out of HR proficiency. RAD51 is an essential gene, protecting cells from genetic instability. Its expression is low and tightly regulated in normal cells and, contrastingly, elevated in a large fraction of cancers, where its level of expression and activity have been linked with sensitivity to genotoxic treatment. In particular, BRCA-deficient tumors show reduced or obliterated RAD51 foci formation and increased sensitivity to platinum salt or PARP inhibitors. However, resistance to treatment sets in rapidly and is frequently based on a complete or partial restoration of RAD51 foci formation. Consequently, RAD51 could be a highly valuable therapeutic target. Here, we review the multiple levels of regulation that impact the transcription of the RAD51 gene, as well as the post-translational modifications that determine its expression level, recruitment on DNA damage sites and the efficient formation of homofilaments. Some of these regulation levels may be targeted and their impact on cancer cell survival discussed.

**Keywords:** RAD51; homologous recombination; DNA repair; genomic instability; DNA break; cancer therapy

## 1. Introduction

DNA damage inflicted by exogenous or endogenous factors such as oxidized radicals, ultra violet (UV) light, ionizing radiation (IR) or genotoxic chemicals are a constant threat to genome integrity. Complex and efficient DNA repair mechanisms preventing the transmission of genetic errors to the progeny are already present in bacteria and have evolved in complexity and specificity in higher eukaryotes. Deficiency in one of the multiple repair mechanisms inevitably results in the accumulation of mutations. DNA repair mechanisms are highly specialized according to the type of damage inflicted. In case of DNA breaks, mechanisms depend on the nature of the break (single or double-strand break), as well as on the cell cycle phase. Unrepaired double-strand breaks (DSB) are highly toxic because they open the door to illegitimate chromosomal rearrangements and can lead

<figure><img src="image_165.png" /></figure>

check for
updates

**Citation:** Orhan, E.; Velazquez, C.; Tabet, I.; Sardet, C.; Theillet, C. *Regulation of RAD51 at the Transcriptional and Functional Levels: What Prospects for Cancer Therapy?* *Cancers* **2021**, *13*, 2930. https://doi.org/10.3390/cancers13122930

Academic Editor: Pierre-Marie Girard

Received: 7 May 2021

Accepted: 8 June 2021

Published: 11 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

<figure><img src="image_166.png" /></figure>

Copyright: © 2021 by the authors. License MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

<footnote n="1">¹ IRCM, Institut de Recherche en Cancérologie de Montpellier U1194 INSERM, Université de Montpellier, 34090 Montpellier, France; esin.orhan@inserm.fr (E.O.); imene.taber@inserm.fr (I.T.); claudesardet@inserm.fr (C.S.)</footnote>

<footnote n="2">² ICM, Institut du Cancer de Montpellier, 34090 Montpellier, France; carolina.velazquez@inserm.fr</footnote>

<footnote>* Correspondence: charles.theillet@inserm.fr</footnote>
to cancer [1]. To keep DNA integrity and stability, cells need to accurately repair these
lesions.

Principal DSB repair mechanisms are non-homologous end joining (NHEJ) repair and homologous recombination (HR) [2]. NHEJ is based on minimal homology of the sequences around the break site in order to ligate the DNA ends. NHEJ is considered an error-prone mechanism that is active throughout the whole cell cycle. During S and G2 phases, HR is activated and competes with NHEJ [3]. Because HR uses the sister chromatid as a template, it is more accurate and avoids misalignment and illegitimate rearrangements that are a risk with NHEJ [4]. Furthermore, DSBs are more susceptible to occurring during the S phase, because of replication fork collapse. For these reasons, HR repair is essential for the integrity of the genome and cell survival. In case of HR deficiency, cells become vulnerable to DSB-induced genome instability [5,6].

When a DSB occurs, the DNA-damage sensors Ataxia-Telangiectasia-Mutated (ATM) and Ataxia Telangiectasia and Rad3-related (ATR) kinases phosphorylate Histone H2AX [7,8]. After this initial signal, DNA repair proteins are recruited to the break site. During the G1 phase of the cell cycle, TP53-Binding Protein (TP53BP1, also called 53BP1) and the shielding complex are recruited at the break site and allow for the recruitment of NHEJ actors, such as DNA-dependent Protein Kinase catalytic subunit (DNA-PKcs), KU70/80, XRCC4 and LIG4 [9,10]. During the S/G2 phases of the cell cycle, Breast Cancer 1 (BRCA1) is phosphorylated and activated by the Cyclin Dependent Kinase 1 (CDK1) [11] and, in the presence of DSB, BRCA1 is also phosphorylated by Checkpoint Kinase 2 (CHK2) that signals the start of the HR repair (HRR) cascade [12]. BRCA1 binds to CtBP-interacting protein (CtIP) that competitively removes 53BP1 from the DSB site and engages HRR [13,14]. CtIP recruits the MRN exonuclease complex which resects the DNA from 5' to 3', leaving long 3' single-strand DNA (ssDNA) overhangs on either side of the DSB [15]. The ssDNA overhangs are rapidly coated and protected by Replication Protein A (RPA). BRCA1 recruits BRCA2 at the break site together with the bridge protein Partner and Localizer of BRCA2 (PALB2). Then, BRCA2 recruits the RAD51 recombinase, which is a crucial protein for the HR to occur. The RAD51 protein forms homopolymeric filaments around the ssDNA and removes RPA. Then, it proceeds to the invasion of the sister chromatid and searches for the matching homologous sequence (Figure 1) [16].

The RAD51 protein is, thus, key for the DNA-strand exchange steps, which HRR is dependent on. RAD51 belongs to the RAD51 protein family that is characterized by a ~230 amino acid domain containing two ATPase motifs called Walker A and Walker B [17]. Human RAD51 actively binds to ssDNA via its N-terminal portion and mediates HR between homologous DNA strands (Figure 2) [18]. RAD51 can be activated by several post-translational modifications, which control its activity at different steps of HRR [19]. We will describe these processes in detail.

RAD51 also plays a role in the repair and re-start of the stalled DNA replication forks, which, in the absence of repair, collapse and leave long stretches of unprotected ssDNA leading to DSB. One of the mechanisms to protect stalled replication forks is replication fork reversal, in which RAD51 is critical [20]. While in normal cells fork reversal is finely tuned to avoid excessive recombination, cancer cells treated with genotoxic agents are challenged with a high number of stalled forks that can overwhelm DNA repair. Thus, elevated levels of RAD51 expression could act as a safeguard to avoid massive fork collapse and protect cancer cells from the accumulation of DNA breaks.

In normal human cells, the expression and activity of RAD51 are tightly controlled in order to avoid aberrant DNA recombination [21]. It is of note that RAD51 expression is strongly increased in breast, thyroid or pancreatic cancer [22-24] and that its overexpression is strongly correlated with poor prognosis [25,26]. Over-activated HRR has been proposed to increase the genetic plasticity of cancer cells due to elevated recombination [21,27]. It is also thought to increase the capacity to repair DNA breaks and, hence, protect cells from DNA-damaging treatments. Indeed, elevated RAD51 expression has been correlated with resistance to chemotherapy [28,29]. Conversely, BRCA-deficient ovarian cancers,
which are HR-deficient, show increased sensitivity to platinum salts and Poly (ADP-ribose) polymerase inhibitors (PARPi) [30]. However, resistance to treatment occurs quickly and, in the case of PARPi, arises mainly by restoring HRR despite BRCA-deficiency. In some studies, restored HRR is monitored by the presence of RAD51 foci in nuclei after treatment-induced DNA damage [31]. This led some authors to propose the use of RAD51 expression or foci detection as a predicting biomarker of HR proficiency to orient the choice of treatments.

<figure><img src="image_167.png" /><figcaption>**Figure 1.** Representation of DNA double-strand break repair choice in the S/G2 phase between homologous recombination non-homologous end joining. The scheme details the steps leading homologous strand invasion and D loop formation. Step 1: 3' end resection by the MRN complex, RPA loading to protect single-strand DNA (ssDNA) from degradation. Step 2: BRCA2 is recruited by BRCA1/PALB2 and loads RAD51 onto the ssDNA overhangs and RPA is dislodged. Step 3: formation of the RAD51 Homopolymeric filament which wind around the ssDNA. Step 4: invasion of the homologous chromatid and search for homology in view of recombination and repair.</figcaption></figure>

The need of alternative treatments for cancer patients that do not respond or develop resistance to DNA-damage-inducing treatments has become evident. RAD51 downregulation has been proposed as a strategy to overcome drug resistance [29]. Thus, better apprehension of RAD51 activity and regulation could lead to new therapeutic strategies for resistant tumors. Several inhibitors of potential RAD51 regulators, either alone or in combination with DNA-damaging agents, have been tested in clinical trials listed in the Supplementary Table (Table S1). In this review, we highlight the importance of RAD51 regulators at the transcriptional and post-translational levels and potential implications for novel treatment strategies. We discuss the clinical significance of RAD51 as a biomarker for HR capacity.
<figure><img src="image_168.png" /><figcaption>**Figure 2.** The schematic view of the RAD51 protein structure. The Walker A and B domains of the RAD51 family are depicted as green and blue boxes. The different interaction and post-translational modification sites are represented.</figcaption></figure>

## 2. Regulation of RAD51 Expression

Transcription factors that regulate RAD51 transcription could, thus, be key in the response of cancer cells to DNA-damaging treatments (Figure 3).

<figure><img src="image_169.png" /><figcaption>**Figure 3.** Identified regulators of RAD51 transcription. The DNA sequences containing the RAD51 promoter and the gene are represented as a purple box and a green box, respectively.</figcaption></figure>

### 2.1. Transcriptional Control of RAD51

#### 2.1.1. Cyclin Dependent Kinases

Cyclin-dependent kinases (CDK) have been proposed to play a role in the transcriptional regulation of RAD51. CDKs are a large family of genes primarily known to regulate cell transition from a cell cycle phase to the next. However, CDK7-13 have been shown to act as key regulators of transcription by way of phosphorylation of the C-terminal domain (CTD) of RNA polymerase II at different serine residues and promote the recruitment of factors necessary for full initiation and elongation of transcripts [32]. During gene transcription by RNA pol II, CDK7, a subunit of TFIIH, phosphorylates Ser5 and activates transcription initiation, whereas CDK9, which forms the pTEF complex with Cyclin T, phosphorylates Ser2 and catalyzes the elongation of the transcripts [33,34]. Interestingly, genetic knockdown or pharmacological inhibition of CDK9 revealed that highly transcribed genes associated with super-enhancers, such as oncogenes or anti-apoptotic genes, were preferentially impacted in cancer cells [34].

Another actively studied CTD Ser2 kinase, whose role was initially discovered in Drosophila, is CDK12 [35]. CDK12, which associates with cyclin K (CCNK), was also shown to regulate transcription elongation. Remarkably, the genetic depletion or pharmacological inhibition of CDK12 appeared to mainly affect long genes comprising multiple exons. This
particularity has been attributed to the fact that CDK12 suppresses intronic polyadenylation, thus allowing for the full-length transcription of these long genes [36–38]. CDK13, which has strong sequence homology with CDK12, has been predicted to share a similar role in pre-mRNA processing [36].

CDK12 and its homolog, CDK13, have been recently proposed to regulate specifically the expression of DNA damage response genes (Table 1) [36,39,40]. Indeed, inhibition of CDK12 either by a drug or by small interfering RNAs (siRNAs) was shown to selectively decrease the expression of DNA damage response genes, such as BRCA1, ATM, FANCD2 and FANCI, possibly because these are large genes with multiple exons [36,39]. Interestingly, Querada and coworkers (2019) suggested that RAD51 expression could be regulated by CDK13, while BRCA1, FANCD2 and ATM transcription was dependent on CDK12 [40]. It thus seems that the knockdown of CDK12/13 affects the transcription of a smaller set of genes compared with the knockdown of CDK9, and it is not yet entirely clear how much both lists overlap. However, the increasing evidence of CDK12 mutations in prostate, triple-negative breast and more so in ovarian cancer has put the focus on this gene that is not considered to belong to the HR pathway per se [41–43]. Indeed, in The Cancer Genome Atlas (TCGA) dataset, 3% of high-grade serous ovarian cancer harbored a CDK12 mutation, an incidence similar to that observed for BRCA1 and BRCA2 (3.5 and 3.2%, respectively) [44]. Furthermore, consistent with a role of CDK12 in the regulation of HR genes, a combination of mechanistic studies with clinical evidence indicated that CDK12-driven transcription of HR genes is crucial for genome stability [45–48]. Hence, CDK12 could be a target of choice to exploit cancer vulnerability associated with DNA damage. Although further understanding of the impact and specificity of CDK inhibition on HR gene expression and activity is needed, it can be hypothesized that the use of selective inhibitors of these kinases could be a strategy of choice to sensitize cancer cells to genotoxic treatment [47]. This is supported by a number of studies on cell lines and preclinical models, showing that PARP-inhibitor (PARP-i) administration combined with a CDK12/13 inhibitor (CDK12-i), such as Dinaciclib or SR-4835, is synergistic compared with PARP-inhibitor alone [40,48–50]. Furthermore, the PARP-i/CDK12-i combination has been suggested to reverse acquired PARP-i resistance [48]. An ongoing clinical trial involving 63 triple-negative breast patients treated with Dinaciclib combined with Velaparib will give us the first clues on the benefits (efficacy and tolerability) of this regimen (Table S1) [50].

### 2.1.2. Member of the E2F Transcription Factor Family

Other documented transcription regulators of RAD51 are E2F family members, which are central gatekeepers of cell cycle progression and survival. Their transcriptional activity is regulated by the binding of retinoblastoma (RB) protein and pocket proteins, p107 and p130 [51,52]. Noticeably, E2F1, E2F4 or E2F7 are activated and stabilized upon DNA damage, possibly by way of phosphorylation by ATM, ATR and CHK2 [53,54]. Elevated E2F1 expression levels have been associated with poor prognosis in non-small cell lung carcinomas and gastrointestinal carcinomas [55,56]. In colon cancer cell models, knockdown of E2F1 was correlated with loss of RAD51 expression and RAD51-dependent DSB repair [57]; furthermore, E2F1 and BRIT1 (MCPH1) were shown to form a complex that activates the transcription of BRCA1, CHK1, p73 and CASP7 [58]. These data strongly suggest that E2F1 could have a role in both activating the transcription of HR genes and their recruitment to DSB.

E2F4 could also play a role in the transcriptional regulation of RAD51. Under hypoxic conditions, E2F4 binds to p130 and represses BRCA1 and RAD51 transcription [59]. The E2F4/p130 driven repression of HR genes could be one of the mechanisms explaining genetic instability in hypoxic cells, a liability that could be exploited to design cancer therapy strategies.
<table><caption>**Table 1.** Regulators of RAD51 at the transcriptional and post-translational levels. Abbreviation: RNA polymerase II (RNA pol II).</caption><thead><tr><th>Regulator of RAD51</th><th>Function</th><th>Level of regulation</th><th>Effect on RAD51</th><th>Drug Target</th></tr></thead><tbody><tr><td>CDK12/CDK13</td><td>RNA pol II regulator</td><td>mRNA expression</td><td>Activator</td><td>Yes</td></tr><tr><td>E2F1</td><td>Transcription Factor</td><td>mRNA expression</td><td>Activator</td><td>No</td></tr><tr><td>E2F4</td><td>Transcription Factor</td><td>mRNA expression</td><td>Repressor</td><td>No</td></tr><tr><td>E2F7</td><td>Transcription Factor</td><td>mRNA expression</td><td>Repressor</td><td>No</td></tr><tr><td>FOXM1</td><td>Transcription Factor</td><td>mRNA expression</td><td>Activator</td><td>No</td></tr><tr><td>EGR1</td><td>Transcription Factor</td><td>mRNA expression</td><td>Activator</td><td>No</td></tr><tr><td>P53</td><td>Transcription Factor</td><td>mRNA expression</td><td>Repressor</td><td>No</td></tr><tr><td>BRD4</td><td>Transcription Activator</td><td>mRNA expression</td><td>Activator</td><td>Yes</td></tr><tr><td>CHD4</td><td>Nucleosome Remodeler</td><td>mRNA expression</td><td>Activator</td><td>No</td></tr><tr><td>PLK1</td><td>Kinase</td><td>Post-translational</td><td>Activator</td><td>Yes</td></tr><tr><td>CK2</td><td>Kinase</td><td>Post-translational</td><td>Activator</td><td>Yes</td></tr><tr><td>CHK1</td><td>Kinase</td><td>Post-translational</td><td>Activator</td><td>Yes</td></tr><tr><td>UCHL3</td><td>Deubiquitinatinase</td><td>Post-translational</td><td>Activator</td><td>No</td></tr><tr><td>FBH1</td><td>Ubiquitin ligase</td><td>Post-translational</td><td>Repressor</td><td>No</td></tr><tr><td>FBX05</td><td>Ubiquitin ligase</td><td>Post-translational</td><td>Repressor</td><td>No</td></tr><tr><td>RFWD3</td><td>Ubiquitin ligase</td><td>Post-translational</td><td>Repressor</td><td>No</td></tr><tr><td>RAD51C</td><td>RAD51 Cofactor</td><td>Homofilament Loading/stabilisation</td><td>Activator</td><td>No</td></tr><tr><td>RAD51B</td><td>RAD51 Cofactor</td><td>Homofilament Loading</td><td>Activator</td><td>No</td></tr><tr><td>RAD51D</td><td>RAD51 Cofactor</td><td>Homofilament Loading</td><td>Activator</td><td>No</td></tr><tr><td>XRCC1</td><td>RAD51 Cofactor</td><td>Homofilament stabilisation</td><td>Activator</td><td>No</td></tr><tr><td>XRCC2</td><td>RAD51 Cofactor</td><td>Homofilament Loading</td><td>Activator</td><td>No</td></tr><tr><td>SWSAP1</td><td>RAD51 Cofactor</td><td>Homofilament stabilisation</td><td>Activator</td><td>No</td></tr><tr><td>PARI</td><td>DNA Helicase</td><td>Homofilament stabilisation</td><td>Repressor</td><td>No</td></tr><tr><td>BLM</td><td>DNA Helicase</td><td>Homofilament stabilisation</td><td>Repressor</td><td>No</td></tr></tbody></table>

E2F7 has been shown to orchestrate transcription under chemotherapy-induced DNA damage. In such conditions, it binds to the promoter of RAD51, BARD1, CtIP, BRIP1 and FANCI and represses their expression. Depletion of E2F7 in U2OS human osteosarcoma cells resulted in increased expression of DNA repair pathway genes, enhanced HRR and replication fork stability [60]. In BRCA2-deficient cells, loss of E2F7 favored resistance to the PARPi Olaparib [61].

Finally, we must mention that CDK2 and CDK4/6 are direct regulators of the transcriptional activity of the E2F-Pocket protein complexes [62,63], adding another, yet unexplored level of complexity to the aforementioned connections between CDKs and HR genes. Taken
together, a better apprehension of the mechanisms governing DNA repair gene transcriptional regulation of the different E2F family members could open novel therapeutic avenues in cancer.

### 2.1.3. Other Transcription Factors

The transcription factor FOXM1 has been suggested to transactivate RAD51 binding directly to its promoter and mediating resistance to chemotherapy [64]. FOXM1 is a pro-oncogenic transcription factor that is overexpressed in glioblastoma multiforme (GBM) and correlated with increased RAD51 expression. FOXM1 inhibition has been proposed as a potential therapeutic strategy in GBM.

Other identified RAD51 transcription regulators comprise EGR1, as an activator, and p53 as a repressor [65,66]. Interestingly, while the normal function of phosphorylated p53 is to repress RAD51 expression, the p53 contact mutant R280K loses this ability and does not reduce DNA-damage-induced RAD51 foci, possibly explaining the resistance to chemotherapy of p53-mutated cancers.

### 2.2. Chromatin-Mediated Regulation of RAD51 Gene Expression

The members of the Bromodomains and Extraterminal (BET) family, comprising 4 genes BRD2, BRD3, BRD4 and BRDt (specific to testis and ovary), are important transcriptional and epigenetic regulators that play key roles in embryogenesis and cancer [67]. They harbor two evolutionary conserved tandem bromodomains (BD1, BD2) and an extraterminal domain (ET) [68]. The BD domains bind to acetyl-lysin residues of DNA-associated proteins, such as histones or transcription factors [69]. The affinity of BRD proteins to multi-acetylated histones is high in general, to the point that BRD4 is considered one of the most abundant proteins on transcriptionally active chromatin. Originally characterized as a key regulator of gene expression during the M to G1 transition [70], BRD4 was found to promote self-renewal of embryonic stem cells [71]. More recently, it was reported to be abundant at enhancer regions and particularly at super-enhancers (SE), which gather several enhancers of highly active genes, including HR genes such as BRCA1 and RAD51 [72,73].

The current model proposes that BRD4 stabilizes transcription factors at enhancers and activates transcription. Through its ET domain, BRD4 binds to various histone modifiers such as the demethylase JMJD6, the methyltransferase NSD3 and the SWIF/SNF enzyme. The recent discovery that super-enhancers in cancer cells are associated with major oncogenes, such as the MYC gene, has identified the BET protein family as a valuable drug target in cancer and fostered the development of several inhibitors (Table 2).

<table><caption>**Table 2.** Molecules targeting potential regulators of RAD51 expression and function tested in clinical trials.</caption><thead><tr><th>Drug</th><th>Target</th><th>Trial phase</th><th>Trial Status</th><th>Results</th><th>Trial Number</th></tr></thead><tbody><tr><td>CYT-0851</td><td>RAD51</td><td>Phase 1/2</td><td>On going</td><td>Not reported</td><td>NCT03997968</td></tr><tr><td>Dinaciclib</td><td>Inhibition of multiple CDKs</td><td>Phase 1</td><td>On going</td><td>Tolerable in combination with veliparib<br>Anti-tumor activity is limited in non-BRCA carriers</td><td>NCT01434316</td></tr><tr><td>INCB054329</td><td>Inhibition of BET proteins</td><td>Phase 1/2</td><td>Completed</td><td>30% Stable disease<br>43% Progressive disease</td><td>NCT02431260</td></tr><tr><td>SYHA1801</td><td>BRD4</td><td>Phase 1</td><td>On going</td><td>Not reported</td><td>NCT04309968</td></tr><tr><td rowspan="2">PLX51107</td><td rowspan="2">BRD4</td><td>Phase 1</td><td>Completed</td><td>Tolerable except for patients with extensive hepatic metastasis</td><td>NCT04022785</td></tr><tr><td>Phase 1</td><td>On going</td><td>Not reported</td><td>NCT04022785</td></tr></tbody></table>
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Drug</th><th>Target</th><th>Trial phase</th><th>Trial Status</th><th>Results</th><th>Trial Number</th></tr></thead><tbody><tr><td>BMS-986158</td><td>Inhibition of BET proteins</td><td>Phase 1</td><td>On going</td><td>Not reported</td><td>NCT03936465</td></tr><tr><td>AZD5153</td><td>BRD4</td><td>Phase 1</td><td>Completed</td><td>Tolerable</td><td>NCT03205176</td></tr><tr><td>GSK525762</td><td>Inhibition of BET proteins</td><td>Phase 1</td><td>Completed</td><td>Manageable tolerability</td><td>NCT01587703</td></tr><tr><td>Volasertib</td><td>PLK1</td><td>Phase 3</td><td>Completed</td><td>Did not meet primary objective in combination with cytarabine</td><td>NCT01721876</td></tr><tr><td>Onvansertib</td><td>PLK1</td><td>Phase 2</td><td>On going</td><td>Not reported</td><td>NCT03414034</td></tr><tr><td>TAK-960</td><td>PLK1</td><td>Phase 1</td><td>Completed</td><td>Terminated due to lack of efficacy</td><td>NCT01179399</td></tr><tr><td>CYC140</td><td>PLK1</td><td>Phase 1</td><td>Completed</td><td>Not reported</td><td>NCT03884829</td></tr><tr><td>NMS-1286937</td><td>PLK1</td><td>Phase 1</td><td>Completed</td><td>Tolerable</td><td>NCT01014429</td></tr><tr><td rowspan="2">CX-4945</td><td rowspan="2">CK2</td><td>Phase 1/2</td><td>Completed</td><td>Not reported</td><td>NCT02128282</td></tr><tr><td>Phase 1/2</td><td>On going</td><td>Not reported</td><td>NCT03904862</td></tr><tr><td>MK-8776</td><td>CHK1</td><td>Phase 2</td><td>Completed</td><td>Similar result as control arm</td><td>NCT01870596</td></tr><tr><td rowspan="2">SRA737</td><td rowspan="2">CHK1</td><td>Phase 1/2</td><td>Completed</td><td>Tolerable</td><td>NCT02797964</td></tr><tr><td>Phase 1/2</td><td>Completed</td><td>Tolerable</td><td>NCT02797977</td></tr><tr><td>LY2603618</td><td>CHK1</td><td>Phase 1/2</td><td>Completed</td><td>Toxicity when combined with pemetrexed+Cisplatin</td><td>NCT01139775</td></tr><tr><td rowspan="2">Prexasertib</td><td rowspan="2">CHK1</td><td>Phase 2</td><td>On going</td><td>Tolerable, only modest activity in BRCA mutant HGSOC patients</td><td>NCT02203513</td></tr><tr><td>Phase 2</td><td>On going</td><td>Not reported</td><td>NCT03414047</td></tr></tbody></table>

However, BRD4 is also an important actor of the mediator complex, which modulates RNA pol II activity, at least in part through the recruitment of the P-TEFb (CCNT1/CDK9 elongation factor. Hence, the inhibition of BRD4 could have effects similar to those of CDK9 inhibition [68,74,75]. Indeed, several studies proposed that the depletion or pharmacological inhibition of BRD4 directly impacted the expression of HR genes, such as RAD51, BRCA1 and BRIP1, and enhanced the sensitivity of cancer cells to PARP inhibitors [72,73]. These results were consolidated by chromatin immunoprecipitation (ChIP) results showing that BRD4-binding to the RAD51 promoter was indeed lost in breast cancer cell lines treated with the BRD4 inhibitor JQ1 and correlated with the loss of RAD51 mRNA expression [73]. These data were corroborated by a study on ovarian cancer cell line models showing that BET inhibitor INCB0544329 synergized with the PARPi Olaparib and resulted in reduced expression of BRCA1 [76]. Altogether, these data suggest that BET inhibitors and more specifically BRD4 inhibitors are interesting leads to induce BRCA-deficiency in cancer cells. Thus, results of ongoing clinical trials using BET-i should be followed with attention.

CHD4 (chromodomain helicase DNA-binding protein 4) is another modifier that has been proposed to impinge on the expression of HR genes. CHD4 is a highly conserved ATPase forming the core subunit of the nucleosome remodeling NuRD complex. NuRL acts both as a transcription repressor and transcription activator and has been shown to play a role in DNA repair [77]. Indeed, CHD4 was shown to be necessary for the recruitment of BRCA1 upon DNA damage in U2OS cells [78,79]. Furthermore, in glioblastoma cells CHD4 was suggested to regulate RAD51 expression directly by binding to the RAD51 promoter and increase the acetylation of histone H3 at lysine 9 (H3K9Ac) [80], possibly
through an interaction with the CBP and p300 co-activators. In line with this model, these acetyl-transferases have been shown to acetylate histones H3 and H4 at the RAD51 and BRCA1 promoters in H1299 human lung cancer cells [81], and CHD4 was shown to interact with p300 in a context of DNA damage [82]. However, it is not yet fully clear whether the CHD4/p300 interaction is critical for RAD51 upregulation or whether it may play a role in the resistance of cancer cells to DNA-damaging treatment.

### 3. Post-Translational Regulation of RAD51

Once RAD51 is transcribed and translated, its functions are orchestrated by post-translational modifications such as phosphorylation and ubiquitination on specific amino acids (Figure 4). A better understanding of these events could give leverage to modulate its activation or stabilization with a potential impact on anti-cancer treatment strategies.

<figure><img src="image_170.png" /><figcaption>**Figure 4.** Post-translational modifications that impinge on RAD51 function. (A) Modifications leading to the activation and loading of RAD51: phosphorylation cascade at Ser14 and Thr13 by PLK1 and CK2, phosphorylation at Thr307/309 by CHK1, ubiquitination at Lys56/57/63 by UCHL3. (B) Modifications leading to the inactivation of RAD51: ubiquitination at Lys58/64 by FBH1, polyubiquitination by FBK05 or RFWD3 leading to the degradation of RAD51 by the proteasome. Blue bubbles (P) represent phosphorylation, red bubbles (Ub) represent ubiquitination.</figcaption></figure>

#### 3.1. Post-Translational Modifications Leading to Activation of RAD51

Phosphorylation by Polo-like kinase 1 (PLK1) and Casein Kinase 2 (CK2): In irradiated HeLa cells, RAD51 is first phosphorylated at Ser14 by PLK1, which primes for the phosphorylation at Thr13 by CK2 [83]. Since RAD51 binding with the FHA domain of NBS1 requires the phosphorylation at both S14 and T13, these sequential phosphorylations could represent important activating steps [83]. Indeed, the RAD51/NBS1 interactions are essential for RAD51 loading onto DSB sites in a BRCA2-independent manner, possibly promoting resistance to IR and PARPi in BRCA2-mutated HeLa cells. It is tempting to speculate that a combined therapy using inhibitors against both PLK1 and PARP or against CK2 and PARP may be an effective approach to improve the prognosis of BRCA-defective cancers.

However, it is unclear whether and through which signals PLK1 and CK2 are activated in response to DSB [84]. PLK1 overexpression has been associated with resistance to doxorubicin, paclitaxel, metformin and gemcitabine via several mechanisms, such as microtubule rearrangement, p53 inactivation or metabolic changes in cell culture and xenograft models [85]. Additionally, the Ser/Thr protein kinase CK2 is constitutively active and ubiquitously expressed, but its role in the DNA damage response is unclear. CK2 is primarily seen as an essential regulator of transcription, including that of major cancer transcription activators, such as MYC or β-catenin. A number of CK2 inhibitors have been described, some of which made it through preclinical stages. CX-4945 (Silmitasertib), a potent ATP competitor with an excellent selectivity, is one of the most promising. It is currently tested in phase I and II clinical trials in mono therapy or in combination
with cisplatin and gemcitabine. The results of these combination trials could be highly
revealing [86], and it should be interesting to investigate the role of RAD51 in the response.

Phosphorylation of RAD51 by CHK1: The CHK1 kinase has been proposed to con-
tribute to the formation of RAD51 foci after genotoxic treatment. Attenuation of CHK1
expression by siRNA transfection was shown to strongly reduce RAD51 foci formation
upon UV light irradiation, while CHK2 inactivation had no effect [87]. RAD51 foci for-
mation depends on its phosphorylation at T307 and T309, which are consensus CHK1
phosphorylation sites. Accordingly, T307-309 phosphorylation and RAD51 foci formation
were reduced in cells treated with a CHK1 inhibitor. The authors determined on human
pancreatic cancer and osteosarcoma cell line models that CHK1 inhibition affected the de
novo formation of RAD51 oligomers but not their stability [88,89]. Interestingly, the binding
and recruitment of RAD51 by BRCA2 appeared to be dependent on its phosphorylation
by CHK1 at T307, giving some mechanistic insight into the impact of CHK1 on RAD51
oligomerization [89]. Taken together, we suggest that the treatment of cancer cells with
CHK1 inhibitor could not only affect the replication checkpoint, but could also interfere
with RAD51 foci formation and, thus, induce HR deficiency. However, most preclinical
and clinical trials have focused on the use of CHK1-i to potentiate replication poisons and
induce catastrophic replication stress [90–92].

Deubiquitination by UCHL3: UCHL3 is a protease of the deubiquitinating enzyme family that catalyzes the removal of ubiquitin moiety from RAD51. RAD51 deubiquitination by UCHL3 was observed both in vitro and in cellulo and shown to be critical for BRCA2-RAD51 interaction, RAD51 recruitment to DSB sites, foci formation, as well as RAD51-mediated D-loop formation [93]. The key role played by UCHL3 in RAD51 activation is illustrated by the dramatic decrease in HR and PARPi sensitivity in UCHL3-depleted cells [93]. Hence, UCHL3 could be a valuable target to induce pharmacological HR deficiency.

3.2. *Post-Translational Modifications Leading to RAD51 Inactivation*

Ubiquitination of RAD51: Several factors control the ubiquitin-dependent RAD51 protein stability. These include two F-box domain protein and an RING-type E3 ligase, which appear to act at different time-points. The F-box DNA helicases FBH1 and FBX05 (also called EMI1) actively assemble SCF (SKP1-CUL1-F-box protein) complexes that act as E3 ubiquitin ligases and target RAD51 for degradation. FBH1 ubiquitinates RAD51 at K58 and K64, resulting in its accumulation in the cytoplasm and a reduction of HRR efficiency after DSB induction [94–98]. FBX05 (EMI1) was the top-hit of an siRNA screen targeting human F-box genes with the aim to identify mediators of olaparib (PARPi) resistance [99]. In this study, FBX05 was the only F-Box protein to co-immunoprecipitate with RAD51. Its silencing dramatically increased steady state levels of the RAD51 protein and induced the accumulation of RAD51 foci [99].

The RING-type E3 ligase RFWD3 binds to and ubiquitinates RPA2 in DNA-damage-
induced foci. It was thus proposed to regulate levels of RPA2 bound to ssDNA [100,101] and
to play a role in HR [99]. It was later shown that RFWD3 also promotes the ubiquitination
of RAD51, reducing its binding to ssDNA and increasing its turnover in DNA damage
foci [102]. Hence, whereas FHB1 and FBX05 seem to act as global regulators of RAD51
protein levels, RFWD3 appeared to be more specifically involved in the regulation of
RAD51 levels bound to ssDNA during the DNA damage or DNA replication stress.

3.3. RAD51 *Loading and Stability at DSB Site by Protein-Protein Interactions*

As described earlier, RAD51 loading at DSB sites and the subsequent RAD51 homopoly
meric-filament assembly, stabilization and timely disassembly are finely tuned steps that
determine HR efficiency (Figure 5). A number of interactors and regulators have been
identified and more might be discovered in the future.
<table><caption>Table 2. *Cont.*</caption><thead><tr><th>Drug</th><th>Target</th><th>Trial phase</th><th>Trial Status</th><th>Results</th><th>Trial Number</th></tr></thead><tbody><tr><td>BMS-986158</td><td>Inhibition of BET proteins</td><td>Phase 1</td><td>On going</td><td>Not reported</td><td>NCT03936465</td></tr><tr><td>AZD5153</td><td>BRD4</td><td>Phase 1</td><td>Completed</td><td>Tolerable</td><td>NCT03205176</td></tr><tr><td>GSK525762</td><td>Inhibition of BET proteins</td><td>Phase 1</td><td>Completed</td><td>Manageable tolerability</td><td>NCT01587703</td></tr><tr><td>Volasertib</td><td>PLK1</td><td>Phase 3</td><td>Completed</td><td>Did not meet primary objective in combination with cytarabine</td><td>NCT01721876</td></tr><tr><td>Onvansertib</td><td>PLK1</td><td>Phase 2</td><td>On going</td><td>Not reported</td><td>NCT03414034</td></tr><tr><td>TAK-960</td><td>PLK1</td><td>Phase 1</td><td>Completed</td><td>Terminated due to lack of efficacy</td><td>NCT01179399</td></tr><tr><td>CYC140</td><td>PLK1</td><td>Phase 1</td><td>Completed</td><td>Not reported</td><td>NCT03884829</td></tr><tr><td>NMS-1286937</td><td>PLK1</td><td>Phase 1</td><td>Completed</td><td>Tolerable</td><td>NCT01014429</td></tr><tr><td rowspan="2">CX-4945</td><td rowspan="2">CK2</td><td>Phase 1/2</td><td>Completed</td><td>Not reported</td><td>NCT02128282</td></tr><tr><td>Phase 1/2</td><td>On going</td><td>Not reported</td><td>NCT03904862</td></tr><tr><td>MK-8776</td><td>CHK1</td><td>Phase 2</td><td>Completed</td><td>Similar result as control arm</td><td>NCT01870596</td></tr><tr><td rowspan="2">SRA737</td><td rowspan="2">CHK1</td><td>Phase 1/2</td><td>Completed</td><td>Tolerable</td><td>NCT02797964</td></tr><tr><td>Phase 1/2</td><td>Completed</td><td>Tolerable</td><td>NCT02797977</td></tr><tr><td rowspan="2">LY2603618</td><td rowspan="2">CHK1</td><td>Phase 1/2</td><td>Completed</td><td>Toxicity when combined with pemetrexed+Cisplatin</td><td>NCT01139775</td></tr><tr><td>Phase 2</td><td>On going</td><td>Tolerable, only modest activity in BRCA mutant HGSOC patients</td><td>NCT02203513</td></tr><tr><td rowspan="2">Prexasertib</td><td rowspan="2">CHK1</td><td>Phase 2</td><td>On going</td><td>Not reported</td><td>NCT03414047</td></tr><tr><td>Phase 2</td><td>On going</td><td>Not reported</td><td>NCT03414047</td></tr></tbody></table>

However, BRD4 is also an important actor of the mediator complex, which modulates RNA pol II activity, at least in part through the recruitment of the P-TEFb (CCNT1/CDK9 elongation factor. Hence, the inhibition of BRD4 could have effects similar to those of CDK9 inhibition [68,74,75]. Indeed, several studies proposed that the depletion or pharmacological inhibition of BRD4 directly impacted the expression of HR genes, such as RAD51 BRCA1 and BRIP1, and enhanced the sensitivity of cancer cells to PARP inhibitors [72,73]. These results were consolidated by chromatin immunoprecipitation (ChIP) results showing that BRD4-binding to the RAD51 promoter was indeed lost in breast cancer cell lines treated with the BRD4 inhibitor JQ1 and correlated with the loss of RAD51 mRNA expression [73]. These data were corroborated by a study on ovarian cancer cell line models showing that BET inhibitor INCB0544329 synergized with the PARPi Olaparib and resulted in reduced expression of BRCA1 [76]. Altogether, these data suggest that BET inhibitors and more specifically BRD4 inhibitors are interesting leads to induce BRCA-deficiency in cancer cells. Thus, results of ongoing clinical trials using BET-i should be followed with attention.

CHD4 (chromodomain helicase DNA-binding protein 4) is another modifier that has been proposed to impinge on the expression of HR genes. CHD4 is a highly conserved ATPase forming the core subunit of the nucleosome remodeling NuRD complex. NuRL acts both as a transcription repressor and transcription activator and has been shown to play a role in DNA repair [77]. Indeed, CHD4 was shown to be necessary for the recruitment of BRCA1 upon DNA damage in U2OS cells [78,79]. Furthermore, in glioblastoma cells CHD4 was suggested to regulate RAD51 expression directly by binding to the RAD51 promoter and increase the acetylation of histone H3 at lysine 9 (H3K9Ac) [80], possibly
through an interaction with the CBP and p300 co-activators. In line with this model, these acetyl-transferases have been shown to acetylate histones H3 and H4 at the RAD51 and BRCA1 promoters in H1299 human lung cancer cells [81], and CHD4 was shown to interact with p300 in a context of DNA damage [82]. However, it is not yet fully clear whether the CHD4/p300 interaction is critical for RAD51 upregulation or whether it may play a role in the resistance of cancer cells to DNA-damaging treatment.

### 3. Post-Translational Regulation of RAD51

Once RAD51 is transcribed and translated, its functions are orchestrated by post-translational modifications such as phosphorylation and ubiquitination on specific amino acids (Figure 4). A better understanding of these events could give leverage to modulate its activation or stabilization with a potential impact on anti-cancer treatment strategies.

<figure><img src="image_171.png" /><figcaption>**Figure 4.** Post-translational modifications that impinge on RAD51 function. (A) Modifications leading to the activation and loading of RAD51: phosphorylation cascade at Ser14 and Thr13 by PLK1 and CK2, phosphorylation at Thr307/309 by CHK1, ubiquitination at Lys56/57/63 by UCHL3. (B) Modifications leading to the inactivation of RAD51: ubiquitination at Lys58/64 by FBH1, polyubiquitination by FBK05 or RFWD3 leading to the degradation of RAD51 by the proteasome. Blue bubbles (P) represent phosphorylation, red bubbles (Ub) represent ubiquitination.</figcaption></figure>

#### 3.1. *Post-Translational Modifications Leading to Activation of RAD51*

Phosphorylation by Polo-like kinase 1 (PLK1) and Casein Kinase 2 (CK2): In irradiated HeLa cells, RAD51 is first phosphorylated at Ser14 by PLK1, which primes for the phosphorylation at Thr13 by CK2 [83]. Since RAD51 binding with the FHA domain of NBS1 requires the phosphorylation at both S14 and T13, these sequential phosphorylations could represent important activating steps [83]. Indeed, the RAD51/NBS1 interactions are essential for RAD51 loading onto DSB sites in a BRCA2-independent manner, possibly promoting resistance to IR and PARPi in BRCA2-mutated HeLa cells. It is tempting to speculate that a combined therapy using inhibitors against both PLK1 and PARP or against CK2 and PARP may be an effective approach to improve the prognosis of BRCA-defective cancers.

However, it is unclear whether and through which signals PLK1 and CK2 are activated in response to DSB [84]. PLK1 overexpression has been associated with resistance to doxorubicin, paclitaxel, metformin and gemcitabine via several mechanisms, such as microtubule rearrangement, p53 inactivation or metabolic changes in cell culture and xenograft models [85]. Additionally, the Ser/Thr protein kinase CK2 is constitutively active and ubiquitously expressed, but its role in the DNA damage response is unclear. CK2 is primarily seen as an essential regulator of transcription, including that of major cancer transcription activators, such as MYC or β-catenin. A number of CK2 inhibitors have been described, some of which made it through preclinical stages. CX-4945 (Silmitasertib), a potent ATP competitor with an excellent selectivity, is one of the most promising. It is currently tested in phase I and II clinical trials in mono therapy or in combination
with cisplatin and gemcitabine. The results of these combination trials could be highly
revealing [86], and it should be interesting to investigate the role of RAD51 in the response.

Phosphorylation of RAD51 by CHK1: The CHK1 kinase has been proposed to con-
tribute to the formation of RAD51 foci after genotoxic treatment. Attenuation of CHK1
expression by siRNA transfection was shown to strongly reduce RAD51 foci formation
upon UV light irradiation, while CHK2 inactivation had no effect [87]. RAD51 foci for-
mation depends on its phosphorylation at T307 and T309, which are consensus CHK1
phosphorylation sites. Accordingly, T307-T309 phosphorylation and RAD51 foci formation
were reduced in cells treated with a CHK1 inhibitor. The authors determined on human
pancreatic cancer and osteosarcoma cell line models that CHK1 inhibition affected the de
novo formation of RAD51 oligomers but not their stability [88,89]. Interestingly, the binding
and recruitment of RAD51 by BRCA2 appeared to be dependent on its phosphorylation
by CHK1 at T307, giving some mechanistic insight into the impact of CHK1 on RAD51
oligomerization [89]. Taken together, we suggest that the treatment of cancer cells with
CHK1 inhibitor could not only affect the replication checkpoint, but could also interfere
with RAD51 foci formation and, thus, induce HR deficiency. However, most preclinical
and clinical trials have focused on the use of CHK1-i to potentiate replication poisons and
induce catastrophic replication stress [90–92].

Deubiquitination by UCHL3: UCHL3 is a protease of the deubiquitinating enzyme family that catalyzes the removal of ubiquitin moiety from RAD51. RAD51 deubiquitination by UCHL3 was observed both in vitro and in cellulo and shown to be critical for BRCA2-RAD51 interaction, RAD51 recruitment to DSB sites, foci formation, as well as RAD51-mediated D-loop formation [93]. The key role played by UCHL3 in RAD51 activation is illustrated by the dramatic decrease in HR and PARPi sensitivity in UCHL3-depleted cells [93]. Hence, UCHL3 could be a valuable target to induce pharmacological HR deficiency.

3.2. *Post-Translational Modifications Leading to RAD51 Inactivation*

Ubiquitination of RAD51: Several factors control the ubiquitin-dependent RAD51 protein stability. These include two F-box domain protein and an RING-type E3 ligase, which appear to act at different time-points. The F-box DNA helicases FBH1 and FBX05 (also called EMI1) actively assemble SCF (SKP1-CUL1-F-box protein) complexes that act as E3 ubiquitin ligases and target RAD51 for degradation. FBH1 ubiquitinates RAD51 at K58 and K64, resulting in its accumulation in the cytoplasm and a reduction of HRR efficiency after DSB induction [94–98]. FBX05 (EMI1) was the top-hit of an siRNA screen targeting human F-box genes with the aim to identify mediators of olaparib (PARPi) resistance [99]. In this study, FBX05 was the only F-Box protein to co-immunoprecipitate with RAD51. Its silencing dramatically increased steady state levels of the RAD51 protein and induced the accumulation of RAD51 foci [99].

The RING-type E3 ligase RFWD3 binds to and ubiquitinates RPA2 in DNA-damage-
induced foci. It was thus proposed to regulate levels of RPA2 bound to ssDNA [100,101] and
to play a role in HR [99]. It was later shown that RFWD3 also promotes the ubiquitination
of RAD51, reducing its binding to ssDNA and increasing its turnover in DNA damage
foci [102]. Hence, whereas FHB1 and FBX05 seem to act as global regulators of RAD51
protein levels, RFWD3 appeared to be more specifically involved in the regulation of
RAD51 levels bound to ssDNA during the DNA damage or DNA replication stress.

3.3. RAD51 Loading and Stability at DSB Site by Protein-Protein Interactions

As described earlier, RAD51 loading at DSB sites and the subsequent RAD51 homopoly
meric-filament assembly, stabilization and timely disassembly are finely tuned steps that
determine HR efficiency (Figure 5). A number of interactors and regulators have been
identified and more might be discovered in the future.
<figure><img src="image_172.png" /><figcaption>**Figure 5.** Intervention of RAD51 cofactors in RAD51 loading and stabilization of the filaments. The BCDX2 intervenes at early stages of RAD51 loading, while CX3 stabilizes the filaments onto the ssDNA.</figcaption></figure>

**RAD51 recruitment:** In an HR-proficient context, RAD51 is actively recruited by a tri-protein complex formed by BRCA1, PALB2 and BRCA2. Of this triad, BRCA2 actively loads RAD51, while the two other partners stabilize the complex. This explains why RAD51 foci formation is reduced or absent in BRCA1 or PALB2 defective cells. However, alternative or safeguard mechanisms have been described to restore RAD51 recruitment in the absence of BRCA1. These range from the loss of expression of 53BP1, the phosphorylation of PALB2 by ATR and the binding of PALB2 by RNF168, which all favor PALB2 activation and accumulation onto DNA damage sites, where it promotes RAD51 recruitment by BRCA2 [103–106]. These observations clearly showed that the intervention of RNF168 could serve as an auxiliary backup mechanism in a BRCA-deficient context [107].

**RAD51 paralogs:** RAD51 is a highly conserved gene going back to recA in bacteria, which evolved during the early stage of eukaryotic evolution into the RAD51 and DMC1 (meiosis specific genes) subfamilies. In higher eukaryotes, the family comprises seven paralogs, RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and the later identified SWSAP1 [108–110].

The RAD51 paralogs share only 20–30% identity at the protein level with RAD51, essentially in the ATP-binding Walker A and B domains and show comparable similarity to each other [109]. The biological functions of RAD51 paralogs have long been questioned. First, answers came from the yeast that possesses two paralogs, rad55 and rad57, which form heterodimers that interact with RAD51 and stimulate strand exchange reactions [111]. In higher eukaryotes, genetic studies on chicken cell lines showed that defective RAD51 paralogs were associated with increased sensitivity to DNA-damaging agents, suggesting a role in HR [111–114]. The importance of the RAD51 paralogs was further underlined by genetic studies showing an increased risk of developing breast and/or ovarian cancer in women bearing RAD51C, RAD51D or XRCC2 mutations [115–117]. This association was confirmed in breast and/or ovarian cancer families and cohorts with no known BRCA1 or BRCA2 mutations, leading to the proposition to include RAD51D and RAD51C to the list of genes screened for breast cancer predisposition [118–120].

In 2001, Masson and coworkers showed that the five human paralogs assembled in two distinct complexes: the first one, BCDX2, is formed of RAD51B, RAD51C, RAD51D and XRCC2, while the second one, identified as CX3, was comprised of RAD51C and XRCC3 [121–123]. More recently, SWSAP1, number six of the gang and non-classical RAD51
paralog, was shown to form a heterodimer with SWS1 termed the Shu complex [124–126]. Whereas the RAD51 paralogs are known to promote RAD51-mediated activities, their precise mechanism of action is not yet completely clear. To decipher their respective roles, several groups have performed conditional knock-out studies of the different genes. Using drug sensitivity and RAD51 foci formation as read-outs, they observed phenotypes of variable severity. The strongest impact was observed for RAD51C and RAD51D disruption, whereas RAD51B knock-out only moderately affected HR [127]. The increased impact of RAD51C and RAD51D was explained by the disruption of the CX3 and BCDX2 complexes and associated destabilization of the XRCC3 and XRCC2 proteins. It was also demonstrated that the CX3 and BCDX2 complexes operate at different stages of HR. Both act downstream of BRCA1 and BRCA2 recruitment to damage sites, but BCDX2 acts upstream of RAD51 recruitment, while CX3 has been suggested to intervene after RAD51 homopolymer formation and possibly stabilize them [123,128]. However, results showing that the CX3 complex binds directly to ssDNA suggest that it could also act prior to RAD51 foci formation [122].

**SWSAP1:** In yeast and worms, disruption of the Shu complex resulted in increased mutagenicity and impaired HR [124,129,130]. Unlike other RAD51 mediators, disruption of the Shu genes primarily impacted on MMS sensitivity and not on other DNA-damaging agents such as IR, hydroxyurea or the Topoisomerase 2 inhibitor etoposide [131]. Consistent with a conserved function of the Shu complex in humans, siRNA depletion of SWS1 and SWSAP1 in human cells resulted in increased MMS sensitivity and reduced RAD51 foci formation [124,125].

**PARI:** PARI is an UvrD-like helicase-domain containing protein that is recruited on DNA by SUMO-modified PCNA and shown to actively reduce RAD51 nucleofilament formation [132]. Indeed, the knock-down of PARI significantly increased RAD51 foci formation in both basal and DNA-damaging conditions [132]. Hence, PARI has been proposed to act as a negative regulator of HR by disassembling toxic RAD51 nucleofilaments. It is, however, not known whether the disassembly of RAD51 filaments by PARI is associated with a post-translational modification of RAD51.

**BLM:** the BLM gene codes for a DNA helicase of the RecQ family, which has been shown to interact with RAD51. RAD51 foci formation appeared constitutively activated in BLM-mutated cells and was suppressed by the ectopic expression of functional BLM, suggesting that BLM negatively regulates RAD51 foci formation [133].

## 4. Pharmacological Inhibition of RAD51

The important role of RAD51 in DNA repair made it an attractive pharmacological target in cancer treatment and has motivated investigators to search for small molecule inhibitors that could disrupt its functions. At least seven compounds antagonizing RAD51 efficiently have been reported.

B02 has been discovered as part of a high-throughput in vitro screening assay scoring for DNA strand exchange. It specifically inhibits human RAD51, but not the E. coli RecA protein [134]. It disrupted RAD51 binding to DNA, inhibits nucleoprotein filament formation and greatly increases the sensitivity of cancer cells to a wide range of genotoxic drugs, including the DNA cross-linking agents cisplatin and mitomycin C, as well as Topoisomerase 1 (Topotecan) and Topoisomerase 2 (doxorubicin) inhibitors. Further studies have shown interesting effects in combination with MAPK inhibitors on melanoma cells or with proton therapy [135,136]. The therapeutic potential of B02 was confirmed in vivo, where it substantially increased the anticancer potential of cisplatin and notably showed little toxic side effects. Indeed, treated mice showed limited weight loss and no macroscopic modifications of the kidney or liver [136]. These results encouraged the development of a new chemical analogue of B02, B17, which presents greater efficacy than B02 in vitro [137]. However, in vivo evaluation has not been reported yet.

Compounds RI-1 and R7a were isolated on the basis of their capacity to inhibit RAD51 binding to ssDNA [138]. Tested on cell cultures, RI-1 showed potent anti-proliferation
activity; however, its short half-life appeared as a strong limit to its in vivo efficacy due to low bio-availability [139].

Compounds IBR2 and derivative IBR120 were isolated on the basis of their capacity to interfere with RAD51 oligomerization and to potentiate its degradation by the proteasome. IBR2 and IBR120 were shown to efficiently inhibit cell growth in a broad set of cancer cell lines [140,141]. In vivo, IBR2 has been tested in single treatment on an imatinib-resistant murine chronic myelogenous leukemia (CML) model, where it significantly prolonged animal survival, without any notable signs of toxicity [140].

Last but not least, the development of compound CYT-0851 is of interest. Firstly, because it is an oral inhibitor that has shown anti-cancer activity on lymphoma as well as the 4T1 mammary tumor in preclinical mouse models [142]. Secondly, because it is, to our knowledge, the only RAD51-i tested in a clinical trial (Table S1).

Altogether, these results suggest that there could be a bright future for small molecule inhibitors of RAD51. However, further tests will be needed to determine in which genetic contexts or tumor types they will prove most effective. Moreover, similarly to other targeted therapies, it is now necessary to identify the best synergistic drug combinations to use with these RAD51 inhibitors. Lastly, induced toxicity and mutagen effects will be critical parameters to take into account for these potent inhibitors.

## 5. Discussion

Due to its prominent role in DNA double-strand break repair, as well as in the protection and repair of stalled replication forks, RAD51 is a crucial actor in the maintenance of genomic stability. Its expression and activity must be tightly controlled in normal cells. Indeed, insufficient levels of RAD51 could cause an accumulation of unrepaired DNA breaks. Conversely, in excess, RAD51 could provoke hyper-recombination and genetic instability. The question about the consequences and advantages of the strong overexpression of RAD51 in a large fraction of tumors at both the RNA and protein levels remains puzzling. As genomic instability is almost inherent in cancer cells, it is tempting to speculate that over-activation of RAD51 could be part of a compensation mechanism to protect cells from excessive genetic instability. Remarkably, RAD51 is strictly essential for cancer cell survival, as illustrated by the 98% cell lines showing elevated dependency scores for RAD51 gene knock-out (the DepMap database www.depmap.org, accessed on 15 March 2021). In comparison, BRCA1 and BRCA2, two important partners of RAD51, are strictly required for the survival of only 84.5 and 84.7% cell lines, respectively (CERES scores below -0.5). It is of note that BRCA1-mutated cell lines are among those showing the highest dependency scores to RAD51. In fact, a number of BRCA1 or BRCA2-deficient cell models do not show complete obliteration of RAD51 foci when exposed to a genotoxic insult, but rather an attenuated response. This can be explained by maintained or restored recruitment of RAD51 onto DNA lesions even in the absence of BRCA1 or BRCA2 due to compensatory mechanisms which are not yet fully understood. Thus, RAD51 is a crucial DNA repair actor in a large majority of cancers, notwithstanding their BRCA proficiency or deficiency. Consequently, it has been shown to be an interesting biomarker, as RAD51 expression levels have been proposed to be potentially predictive of resistance to treatment [143,144]. Additionally, assays aimed at determining the capacity of tumor cells to form RAD51 foci when challenged with a DNA-damaging treatment have been proposed as biomarkers of response to PARP-i (NCT04780945) [145]. Furthermore, RAD51 appears as a valuable therapeutic target, especially in tumors prone to accumulate DNA breaks, such as cancers showing strongly rearranged genomes. The design of pharmacological approaches that would efficiently impair or attenuate RAD51 recruitment onto DNA lesions and/or the formation of presynaptic filaments is, in our opinion, the therapeutic strategy of choice, especially in combination with DNA-break-inducing agents. As described above, approaches are aiming at downregulating the mRNA expression of genes belonging to the HR pathway, among which RAD51 is being actively studied. Given the central role of RAD51 in HR, inhibitors that directly target the protein are, as discussed above, interesting
leads to be followed (Table S1) [146,147]. Moreover, the future may also bring about further approaches based on inhibitors targeting either enzymes that activate RAD51 or target its cofactors and interfere with homopolymeric filament formation or stabilization. Which approach will be most effective remains undetermined, but we are convinced that targeting RAD51 directly or its cofactors will have great clinical potential in the future.

**Supplementary Materials:** The following is available online at https://www.mdpi.com/article/10.3390/cancers13122930/s1, Table S1: Clinical Trials.

**Author Contributions:** E.O. conceptualized the overall idea for this review, organized the thoughts and designed the backbone of this review. E.O. and C.T. wrote this review. I.T., C.V. and C.S. reviewed and edited this review. All authors have read and agreed to the published version of the manuscript.

**Funding:** E.O. is currently a doctoral fellow of the Labex Epigenmed. Furthermore, the authors wish to express their gratitude for the support of the SIRIC Montpellier Cancer Grant INCa-DGOS-Inserm_12553 to E.O., I.T. and C.V.

**Acknowledgments:** Figures are created with BioRender.com.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Abbreviations**

<table><tr><td>ATM</td><td>Ataxia-Telangiectasia Mutated</td></tr><tr><td>ATR</td><td>Ataxia Telangiectasia and Rad3 Related</td></tr><tr><td>BD</td><td>Bromodomain</td></tr><tr><td>BET</td><td>Bromodomains and Extraterminal</td></tr><tr><td>BRCA1</td><td>Breast Cancer 1</td></tr><tr><td>BRCT</td><td>BRCA1 Carboxy-Terminus</td></tr><tr><td>CCNK</td><td>Cyclin K</td></tr><tr><td>CDK</td><td>Cyclin-Dependent Kinase</td></tr><tr><td>ChIP</td><td>Chromatin Immunoprecipitation</td></tr><tr><td>CHD4</td><td>Chromodomain Helicase DNA-binding Protein 4</td></tr><tr><td>CK2</td><td>Casein Kinase 2</td></tr><tr><td>CML</td><td>Chronic Myelogenous Leukemia</td></tr><tr><td>CTD</td><td>C-Terminal Domain</td></tr><tr><td>CtIP</td><td>CtBP-Interacting Protein</td></tr><tr><td>DNA</td><td>Deoxyribonucleic acid (necessary?)</td></tr><tr><td>DNA-PKcs</td><td>DNA-dependent Protein Kinase Catalytic Subunit</td></tr><tr><td>DSB</td><td>Double-Strand Break</td></tr><tr><td>ET</td><td>Extraterminal Domain</td></tr><tr><td>GBM</td><td>Glioblastoma Multiforme</td></tr><tr><td>H2AX</td><td>Histone 2AX</td></tr><tr><td>HR</td><td>Homologous Recombination</td></tr><tr><td>HRR</td><td>Homologous Recombination Repair</td></tr><tr><td>H3K9Ac</td><td>Histone H3 at lysine 9</td></tr><tr><td>IR</td><td>Ionizing Radiation</td></tr><tr><td>NHEJ</td><td>Non-Homologous End Joining</td></tr><tr><td>PALB2</td><td>Partner and Localizer of BRCA2</td></tr><tr><td>PARPi</td><td>Poly (ADP-ribose) Polymerase Inhibitors</td></tr><tr><td>PLK1</td><td>Polo-like kinase 1</td></tr><tr><td>RB</td><td>Retinoblastoma</td></tr><tr><td>RPA</td><td>Replication Protein A</td></tr><tr><td>SE</td><td>Super-Enhancers</td></tr><tr><td>ssDNA</td><td>Single-Strand DNA</td></tr><tr><td>TCGA</td><td>The Cancer Genome Atlas</td></tr><tr><td>UV</td><td>Ultra-violet</td></tr><tr><td>5MBP1</td><td>TP53-binding protein</td></tr></table>
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