Wwox Binding to the Murine Brca1-BRCT Domain Regulates Timing of Brip1 and CtIP Phospho-Protein Interactions with This Domain at DNA Double-Strand Breaks, and Repair Pathway Choice

Wwox-deficient human cells show elevated homologous recombination, leading to resistance to killing by double-strand break-inducing agents. Human Wwox binds to the Brca1 981-PPLF-984 Wwox-binding motif, likely blocking the pChk2 phosphorylation site at Brca1-S988. This phosphorylation site is conserved across mammalian species; the PPLF motif is conserved in primates but not in rodents. We now show that murine Wwox does not bind Brca1 near the conserved mouse Brca1 phospho-S971 site, leaving it open for Chk2 phosphorylation and Brca1 activation. Instead, murine Wwox binds to Brca1 through its BRCT domain, where pAbraxas, pBrip1, and pCtIP, of the A, B, and C binding complexes, interact to regulate double-strand break repair pathway response. In Wwox-deficient mouse cells, the Brca1-BRCT domain is thus accessible for immediate binding of these phospho-proteins. We confirm elevated homologous recombination in Wwox-silenced murine cells, as in human cells. Wwox-deficient murine cells showed increased ionizing radiation-induced Abraxas, Brca1, and CtIP foci and long resected single-strand DNA, early after ionizing radiation. Wwox deletion increased the basal level of Brca1-CtIP interaction and the expression level of the MRN-CtIP protein complex, key players in end-resection, and facilitated Brca1 release from foci. Inhibition of phospho-Chk2 phosphorylation of Brca1-S971 delays the end-resection; the delay of premature end-resection by combining Chk2 inhibition with ionizing radiation or carboplatin treatment restored ionizing radiation and platinum sensitivity in Wwox-deficient murine cells, as in human cells, supporting the use of murine in vitro and in vivo models in preclinical cancer treatment research.


Introduction
Expression of the Wwox tumor suppressor protein, with highly conserved amino acid sequences across species from fish to man [1], is frequently downregulated in human and mouse cancer genomes through deletion, translocation, and promoter hypermethylation, of aa S988, which is phosphorylated by pChk2, and is critical for DSB repair pathway choice [16,38]. To determine if this exon 11 Wwox-binding motif is conserved in mouse Brca1, we aligned Brca1 aa sequences of different vertebrate species. The 981 PPLF 984 Brca1 sequence is conserved in primates but not in rodent species, though the mouse pChk2 phosphorylation site at aa S971 is conserved ( Figure 1A) (canine and bovine species also do not show a known Wwox binding motif in this position, not shown). To test for Wwox-Brca1 interaction in mouse cells, WT MEFs were transiently transfected with Myc-Wwox, interacting proteins pulled down using Myc beads, and immunoblotted with Brca1 antiserum. Despite the lack of the PPLF motif, the Myc-Wwox protein co-immunoprecipitated Brca1, both unphospho-and hyperphosphorylated forms ( Figure 1B). To determine where Wwox binds in mouse Brca1, we performed co-immunoprecipitation (co-IP) using MEF cells expressing mutant Brca1. Although murine Brca1 exon 11 does not exhibit a Wwoxbinding motif, to rule out the possibility that Wwox binds through exon11, we tested Wwox-Brca1 interaction in mouse MEFs with exon 11 deleted (Brca1 ∆ex11/∆ex11 ) [39]. The mutant Brca1 ∆ex11 binds to Myc-Wwox, confirming that the exon 11 region is dispensable for the mouse Brca1-Wwox interaction ( Figure 1C). Next, to consider a contribution of the Brca1-BRCT repeats to Wwox binding, we performed co-IP experiments, using MEFs expressing a cancer-associated missense variant M1717R (Brca1 M1717R/M1717R , equivalent to human Brca1 M1775R) located in the BRCT domain, that disrupts Brca1-BRCT complex formation with pAbraxas, pBrip1, and pCtIP [28,34]. WT Brca1 efficiently interacted with Myc-Wwox, whereas the Brca1-BRCT M1717R mutant protein did not ( Figure 1D). Furthermore, in situ association of Wwox-Brca1 is markedly decreased in another BRCT domain mutant cell line, Brca1 S1655F/S1655F , as measured by PLA (Supplementary Figure S1), indicating that the WT mouse Brca1-BRCT tandem domain is required for Wwox-Brca1 interaction and this interaction is eliminated by these point mutations.

Wwox Interaction with Brca1-BRCT Is Independent of Binding of Abraxas, Brip1, and CtIP
It is known that Brca1 is involved in HR and that its C-terminal tBRCT domain directly interacts with pAbraxas, pBrip1, and pCtIP. These three phospho-proteins participate in specific, timed steps in HR repair, while contributing to the stability of the genome [19,40]. Because the BRCT domain of Brca1 with the point mutations M1717R or S1655F is unable to bind the A, B, C complex proteins, it is likely that these mutations cause major disruption in the folded structure of the tBRCT domain [28,40]. Since our result suggests that Wwox binds to the tBRCT domain of Brca1 in mouse cells, we considered two possibilities, direct Brca1-BRCT-Wwox binding or indirect binding through Wwox-interaction with one of the Abraxas, Brip1, and/or CtIP proteins. Thus, we examined the aa sequence of mouse and human Brca1-BRCT domains for potential Wwox-binding motifs [41]. We did not find a known Wwox interaction motif in the murine or human Brca1-BRCT domain ( Figure 1F). However, the evolutionarily highly conserved Wwox gene encodes a ubiquitous protein participating in a myriad of important cellular pathways to which literally hundreds of direct and potentially indirect binding partners have been described, with mechanisms and interaction motifs of the majority not defined [41]. To determine if Wwox directly binds to the tBRCT domain, not through interaction with Abraxas, Brip1, CtIP proteins, we performed Wwox-Brca1 co-IP in triple A, B, C mutant MEFs (Abraxas S404A/S404A ; Brip1 S994A/S994A ; CtIP S326A/S326A ), referred to as ABC mutant cells [34]. In ABC mutant cells, none of the Abraxas, Brip1, or CtIP proteins are able to interact with Brca1, due to abrogation of their phosphorylation sites [33,34,42]. Therefore, if one of the Abraxas, Brip1, or CtIP proteins function as a linker between Wwox and Brca1, Wwox-Brca1 interaction would be disrupted in ABC mutant cells. Instead, we observed that, in the ABC mutant cells, Wwox protein does bind to the Brca1-BRCT domain ( Figure 1E), suggesting that the A, B, and C complex formation through Brca1-BRCT domain is unnecessary for Wwox-Brca1-BRCT interaction in mouse cells. Amino acid sequence alignment of Brca1 loci of primate and rodent species near mouse S971 and human S988 in exon 11, serines critical for activation of the HR repair pathway conserved across mammalian species; the PPLF motif, the primate Wwox-binding site, is not conserved in rodent Brca1. (B) Transfected Myc-Wwox co-immunoprecipitated with mouse Brca1. Transfected Myc-Wwox in MEFs pulled down both non-phospho and IR induced-phospho-Brca1. Note: Brca1 upshift after IR. (C) Brca1 exon 11, which is essential for Wwox-Brca1 interaction in human cells, is dispensable for Wwox-Brca1 interaction in MEFs. Both WT and Δexon11 (Δex11) Brca1 co-immunoprecipitated with Myc-Wwox in MEFs. (D) Brca1 BRCT domain mutant M1717R (MR) failed to interact with Wwox. E is empty vector transfected negative control. (E) Interaction of the pAbraxas, pBrip1, and pCtIP with BRCT domain in Brca1 is dispensable for Wwox-Brca1 binding, as shown by Wwox binding in ABC mutant cells, in which those three phospho-ligands do not bind the BRCT domain. (F) The human and mouse Brca1 BRCT domains are aligned, with sites of the SF and MR mutants that prevent binding of the pAbraxas, pBrip1, and pCtIP proteins marked in green. No canonical Wwox binding motif is observed in the mouse or human Brca1 BRCT region.

Brca1 Independent Interaction of Wwox with Abraxas, Brip1 and CtIP
As we considered whether Wwox associated with a ligand that binds to the Brca1-BRCT domain, we performed co-IPs of Wwox with Abraxas, Brip1, and CtIP. To examine the Wwox-Abraxas interaction, we transiently transfected mouse HA-Abraxas into WT MEFs, pulled-down HA-Abraxas with HA magnetic beads, immunoblotted with HA and Wwox antisera, and observed that mouse Abraxas co-immunoprecipitated with Wwox in MEFs, and IR treatment did not affect the binding level ( Figure 2A). We then tested for a Brip1-Wwox interaction in Brip1 FHWT/FHWT MEFs [40] expressing FLAG-HA tagged Brip1 (FH-Brip1, given lack of a suitable Brip1 antiserum for MEFs). Interestingly, co-IP showed binding of Wwox to Brip1, with the interaction possibly enhanced by IR treatment ( Figure  2B). Recently, a role for Brip1 in end-resection in cooperation with CtIP was reported in

Brca1 Independent Interaction of Wwox with Abraxas, Brip1 and CtIP
As we considered whether Wwox associated with a ligand that binds to the Brca1-BRCT domain, we performed co-IPs of Wwox with Abraxas, Brip1, and CtIP. To examine the Wwox-Abraxas interaction, we transiently transfected mouse HA-Abraxas into WT MEFs, pulled-down HA-Abraxas with HA magnetic beads, immunoblotted with HA and Wwox antisera, and observed that mouse Abraxas co-immunoprecipitated with Wwox in MEFs, and IR treatment did not affect the binding level ( Figure 2A). We then tested for a Brip1-Wwox interaction in Brip1 FHWT/FHWT MEFs [40] expressing FLAG-HA tagged Brip1 (FH-Brip1, given lack of a suitable Brip1 antiserum for MEFs). Interestingly, co-IP showed binding of Wwox to Brip1, with the interaction possibly enhanced by IR treatment ( Figure 2B). Recently, a role for Brip1 in end-resection in cooperation with CtIP was reported in which Brip1-CtIP interaction was observed only after inducing DSBs [43]. In our mouse models, Brip1 interacts with CtIP with and without IR ( Figure 2B). We also observed Wwox-CtIP interaction in WT MEFs ( Figure 2C). To determine if Wwox-Brip1/CtIP binding is due to Brca1-BRCT interaction with Brip1/CtIP, we tested Wwox-Brip1 binding in Brca1silenced FH-Brip1 MEFs ( Figure 2D) and Wwox-CtIP binding in Bard1 −/− ; 53BP1 −/− MEFs ( Figure 2E). Brca1 and Bard1 form a heterodimeric complex, and thus Brca1 stability in cells lacking Bard1 is dramatically reduced [44]. Our IP results showed that Wwox-Brip1/CtIP interactions are independent of Brca1, suggesting those three proteins are in a complex. In addition, Wwox binding to Abraxas, Brip1 and CtIP proteins, as well as to the Brca1-BRCT domain, may cooperatively regulate Brca1-A, B, C complex formation, possibly by blocking their phosphorylation. which Brip1-CtIP interaction was observed only after inducing DSBs [43]. In our mouse models, Brip1 interacts with CtIP with and without IR ( Figure 2B). We also observed Wwox-CtIP interaction in WT MEFs ( Figure 2C). To determine if Wwox-Brip1/CtIP binding is due to Brca1-BRCT interaction with Brip1/CtIP, we tested Wwox-Brip1 binding in Brca1-silenced FH-Brip1 MEFs ( Figure 2D) and Wwox-CtIP binding in Bard1 −/− ; 53BP1 −/− MEFs ( Figure 2E). Brca1 and Bard1 form a heterodimeric complex, and thus Brca1 stability in cells lacking Bard1 is dramatically reduced [44]. Our IP results showed that Wwox-Brip1/CtIP interactions are independent of Brca1, suggesting those three proteins are in a complex. In addition, Wwox binding to Abraxas, Brip1 and CtIP proteins, as well as to the Brca1-BRCT domain, may cooperatively regulate Brca1-A, B, C complex formation, possibly by blocking their phosphorylation.

Effect of Wwox Loss on Timing of Appearance of Abraxas, Brca1 and CtIP Proteins at IR-Induced DSB Foci
The Brca1 tandem BRCT repeat domain forms a groove as a mutually exclusive binding site for pAbraxas, pBrip1, or pCtIP [27,45]. These Brca1 binding complexes are designated Brca1-BRCT A, B and C and are involved in the Brca1 repair pathway downstream functions [20,21,24,40]. We wondered if Wwox binding to the Brca1-BRCT domain or to A, B, C proteins themselves could influence recruitment of Abraxas, Brca1, or CtIP proteins to DSBs. To determine if the absence of Wwox protein expression in the Wwox KO MEF cells affects the timing of appearance of the Brca1-associated complex proteins that control execution of HR repair functions at IR-induced DSBs, we performed time-course studies to quantify Abraxas, Brca1, and CtIP foci at the DSBs in WT vs. KO MEFs after 10 γ IR.
As shown in Figure

Effect of Wwox Loss on Timing of Appearance of Abraxas, Brca1 and CtIP Proteins at IR-Induced DSB Foci
The Brca1 tandem BRCT repeat domain forms a groove as a mutually exclusive binding site for pAbraxas, pBrip1, or pCtIP [27,45]. These Brca1 binding complexes are designated Brca1-BRCT A, B and C and are involved in the Brca1 repair pathway downstream functions [20,21,24,40]. We wondered if Wwox binding to the Brca1-BRCT domain or to A, B, C proteins themselves could influence recruitment of Abraxas, Brca1, or CtIP proteins to DSBs. To determine if the absence of Wwox protein expression in the Wwox KO MEF cells affects the timing of appearance of the Brca1-associated complex proteins that control execution of HR repair functions at IR-induced DSBs, we performed time-course studies to quantify Abraxas, Brca1, and CtIP foci at the DSBs in WT vs. KO MEFs after 10 γ IR.
As shown in Figure Figure 3D). We also compared complexed Abraxas-Brca1 proteins at DSBs using Brca1 FHWT/FHWT cells [46], with or without Wwox silencing. In siControl transfected cells, the number of Abraxas-Brca1 co-localized foci was increased at 0.5 h, reached a peak at 1 h, and was decreased at 2 h after IR. In Wwox-silenced cells, the number of co-localized foci reached a peak at 0.5 h and decreased over time ( Figure 3E, 0.5 h post-IR representative image shown in Figure 3F). Our results imply that Wwox binding to Brca1-BRCT domain proteins delays Abraxas-mediated Brca1 recruitment to foci. Therefore, loss of Wwox facilitates Abraxas-mediated Brca1 recruitment to DSBs. MRN complex (Mre11-Rad50-NBS1) and CtIP are crucial for promoting resection activity at the point of commitment to the HR repair pathway [23,[30][31][32]. As Wwox expression has a role in attenuating the end-resection process, we asked if Wwox could modulate the level of MRN/CtIP complex proteins that interact with mouse Brca1. Thus, we silenced Wwox expression in three different mouse cell lines and measured the steady-state levels of CtIP and individual components of the MRN complex. As shown in Figure 4D, Wwox knockdown upregulated the basal levels of Brca1-C complex proteins vs. the siControl transfected cells, implying that Wwox regulates steady-state levels of complex C proteins ( Figure 4D). Since these proteins are required for end-resection during HR repair, our data support the proposal that Wwox modulates the timing of recruitment of the C complex Error bars representing S.E.M and p-values were calculated by unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001); N.S., not significant. Experiments were carried out using two different wild-type (WT3, WT4) and KO (KO3, KO5) clones in duplicate.
Brca1-mediated HR repair is initiated by DNA end-resection at DSBs, which generates 3 ssDNA that can invade the homologous DNA strand. Thus, end-resection is a critical point for DSB repair pathway choice. It has been reported that CtIP is a key protein that induces a DNA nick to initiate end-resection by the MRN complex (Mre11-Rad50-NBS1) [23,[30][31][32]47]. We compared CtIP focus formation at early time points after IR treatment in Wwox WT vs. KO MEFs. The percentage of CtIP positive cells with >5 foci in KO cells exceeded those of WT cells at 0.5 h post-IR ( Figure 3G, 1 h post-IR representative image shown in Figure 3H). To examine CtIP protein at DSBs, we performed a PLA assay using CtIP and γH2AX antisera. Consistent with the IF results, we observed a dramatic increase of PLA foci at 0.5 h post-IR in Wwox KO cells (Supplementary Figure S2). In WT MEFs, the number of PLA foci gradually increased in a time-dependent manner (Supplementary Figure S2).

Loss of Wwox Expression in Mouse Cells Supports Initiation of HR through Rapid Engagement of End-Resection
To determine if the absence of Wwox expression influences DNA end-resection, an essential early step for the HR pathway of DSB repair, we measured resection lengths at the level of individual DNA fibers by SMART assay in WT and KO MEFs ( Figure 4A). ssDNA lengths at 1 h were significantly longer in IR-treated KO than WT MEFs, with long resected ssDNAs (5-10 µm) constituting~50% of measured fibers, while ssDNAs are still short in WT cells (1-2 µm) ( Figure 4A), indicating that Wwox absence accelerates the DNA end-resection process. Consistent with the SMART assay result, IR induced focus formation of pRPA32-S4/S8, a ssDNA marker, was elevated in Wwox KO vs. WT cells at 0.5 h post-IR ( Figure 4B). In addition, pRPA recruitment to chromatin fraction was dramatically increased in Wwox KO cells after IR treatment ( Figure 4C).
MRN complex (Mre11-Rad50-NBS1) and CtIP are crucial for promoting resection activity at the point of commitment to the HR repair pathway [23,[30][31][32]. As Wwox expression has a role in attenuating the end-resection process, we asked if Wwox could modulate the level of MRN/CtIP complex proteins that interact with mouse Brca1. Thus, we silenced Wwox expression in three different mouse cell lines and measured the steady-state levels of CtIP and individual components of the MRN complex. As shown in Figure 4D, Wwox knockdown upregulated the basal levels of Brca1-C complex proteins vs. the siControl transfected cells, implying that Wwox regulates steady-state levels of complex C proteins ( Figure 4D). Since these proteins are required for end-resection during HR repair, our data support the proposal that Wwox modulates the timing of recruitment of the C complex proteins to DSBs following IR exposure.
Next, to determine if the elevated end-resection in Wwox deficient cells affects the efficiency of HR, we employed HR repair pathway reporter assays. In these assays, HR efficiency is quantified by expression of a GFP gene that has been correctly repaired via HR following induction of DSB at an ISce1 site. We transfected MEFs carrying this construct with siControl or siWwox oligonucleotides. As shown in Figure 4E, in Wwox-silenced cell clones, the absence of Wwox protein enhanced HR activity vs. siControl clones, which is consistent with our previous observation in human reporter cells [7]. We also assessed Rad51 focus formation by IF detection of foci following IR. Both WT and Wwox KO cells exhibit some RAD51 foci without IR treatment, with more Rad 51 foci in the KO vs. WT cells, suggesting that the Wwox absence affects basal activity of the HR repair pathway. Within 1 h of IR treatment, Rad51 foci were significantly increased in Wwox-deficient clones, with twice as many cells displaying >10 foci vs. WT clones. Unlike WT MEFs, the percentage of KO cells with >10 foci was slightly decreased by 2 h. Rad51 foci then gradually increased in both WT and KO cells and concordantly reached their peak levels by 4 h post-IR ( Figure 4F). This elevated number of Rad51 positive cells upon IR treatment is also observed in Wwox silenced MEFs (Supplementary Figure S3). as a marker of mutant accumulation in the Wwox-deficient cells. The mutation frequency was increased in IR-treated KO MEFs, with WT cells acquiring 3.5-fold fewer colonies with HPRT mutations than KO cells ( Figure 4G), likely due to inappropriate repair pathway choice. Results of these experiments collectively confirm that Wwox loss dysregulates DSB repair pathway choice after DSB induction by promoting activation of the HR pathway through immediate end-resection post-IR.  Survival data for the two WT and two KO clones are presented as averages. Error bars represent S.E.M and p-values were calculated by an unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001); N.S., not significant.
Since our initial observations in MEFs established a genome caretaker function for Wwox [7], we next investigated whether Wwox could suppress mutation acquisition by assessment of colonies surviving in 6TG supplemented medium due to HPRT mutation, as a marker of mutant accumulation in the Wwox-deficient cells. The mutation frequency was increased in IR-treated KO MEFs, with WT cells acquiring 3.5-fold fewer colonies with HPRT mutations than KO cells ( Figure 4G), likely due to inappropriate repair pathway choice. Results of these experiments collectively confirm that Wwox loss dysregulates DSB repair pathway choice after DSB induction by promoting activation of the HR pathway through immediate end-resection post-IR.

Inhibition of End-Resection by Chk2 Silencing Sensitizes Wwox KO Cells to DNA Damaging Agents
Initiation of HR and SSA pathway choice is dependent on DNA end-resection. Our previous study found that Wwox deficiency in human cells treated with IR resulted in early DNA end-resection to tip the initial repair pathway choice toward HR and SSA, leading to resistance to DSB-inducing agents, IR, and cis-or carboplatin [7].
It is well documented that Brca1 activation requires its phosphorylation by ATM/ATR and pChk2 [14,37]. Blocking pChk2 phosphorylation of Brca1 at S988 in human, S971 in mouse, and delayed end-resection at DSBs [38]. Thus, we hypothesized that delay of the early occurring end-resection in Wwox KO cells by inhibition of Brca1 phosphorylation by Chk2 could sensitize the Wwox KO cells to DNA damaging agents. To test this idea, we pretreated cells with Chk2 selective inhibitor, BML-277, or silenced Chk2 expression before IR or carboplatin treatment, and measured sensitivity by clonogenicity assays. Pretreatment with BML-277 dramatically reduced relative survival of Wwox KO cells after IR ( Figure 5A). Similarly, Chk2 silenced Wwox KO MEFs were more sensitive to carboplatin treatment ( Figure 5B). However, both Chk2 inhibitor treatment and Chk2 silencing did not impact relative survival in WT cells ( Figure 5A,B). This selective killing effect on Wwox-deficient cells by combination treatment of IR/Carboplatin and Chk2 inhibitor was also observed in human cells [10], suggesting that inhibition or delay of Brca1 phosphorylation at S971 (S988 in human) by Chk2 inhibitor sensitize Wwox negative cells. Thus, concurrent treatment of IR/carboplatin with Chk2 inhibitor could be an alternative therapy to selectively target the high percentage of Wwox negative, treatment resistant tumors.
GFP, in cells silenced for Wwox expression. (F) IR-induced Rad51 foci are increased in Wwox KO MEFs. Experiments were carried out using two different wild-type (WT3, WT4) and KO (KO3, KO5) clones in duplicate. (G) DSB repair in Wwox negative cells increases mutation acquisition. Frequency of mutations of the HPRT gene is increased in Wwox KO vs. wild-type WT MEFs following IR exposure. Survival data for the two WT and two KO clones are presented as averages. Error bars represent S.E.M and p-values were calculated by an unpaired t-test (* p < 0.05, ** p < 0.01, *** p < 0.001); N.S., not significant.

Inhibition of End-Resection by Chk2 Silencing Sensitizes Wwox KO Cells to DNA Damaging Agents
Initiation of HR and SSA pathway choice is dependent on DNA end-resection. Our previous study found that Wwox deficiency in human cells treated with IR resulted in early DNA end-resection to tip the initial repair pathway choice toward HR and SSA, leading to resistance to DSB-inducing agents, IR, and cis-or carboplatin [7].
It is well documented that Brca1 activation requires its phosphorylation by ATM/ATR and pChk2 [14,37]. Blocking pChk2 phosphorylation of Brca1 at S988 in human, S971 in mouse, and delayed end-resection at DSBs [38]. Thus, we hypothesized that delay of the early occurring end-resection in Wwox KO cells by inhibition of Brca1 phosphorylation by Chk2 could sensitize the Wwox KO cells to DNA damaging agents. To test this idea, we pretreated cells with Chk2 selective inhibitor, BML-277, or silenced Chk2 expression before IR or carboplatin treatment, and measured sensitivity by clonogenicity assays. Pretreatment with BML-277 dramatically reduced relative survival of Wwox KO cells after IR ( Figure 5A). Similarly, Chk2 silenced Wwox KO MEFs were more sensitive to carboplatin treatment ( Figure 5B). However, both Chk2 inhibitor treatment and Chk2 silencing did not impact relative survival in WT cells ( Figure 5A,B). This selective killing effect on Wwox-deficient cells by combination treatment of IR/Carboplatin and Chk2 inhibitor was also observed in human cells [10], suggesting that inhibition or delay of Brca1 phosphorylation at S971 (S988 in human) by Chk2 inhibitor sensitize Wwox negative cells. Thus, concurrent treatment of IR/carboplatin with Chk2 inhibitor could be an alternative therapy to selectively target the high percentage of Wwox negative, treatment resistant tumors.

Discussion
In our initial study of Wwox in human breast cancer cells [7], we reported Wwox interaction with Brca1 and upregulated HR/SSA pathways in Wwox-deficient cells. We also noted that established mouse KO MEFs and Wwox-deficient normal breast cells responded to IR or cisplatin exposure similarly to human cancer cells, and showed resistance to killing relative to WT MEFs and Wwox-proficient breast cells, respectively [6,7]. Our current study of the role of murine Wwox in response to repair of DSBs induced by IR or platinum drugs has found that: Wwox interacts with the Brca1-BRCT domain; Wwox also binds to Abraxas, Brip1, and CtIP, independently of Brca1, suggesting two levels of Wwox regulation of Brca1-BRCT complex formation; Wwox-deficient murine cells showed increased IR-induced Abraxas, Brca1, and CtIP foci formation and premature long resected single-strand DNA, early after IR; Wwox deletion increased the basal level of Brca1-CtIP interaction and expression level of MRN-CtIP protein complex; the delay of premature end-resection by combining Chk2 inhibition with IR or carboplatin treatment successfully restored IR and platinum sensitivity in Wwox deficient cells in vitro.
Since mouse models carrying knockout genes for numerous tumor suppressor genes (including Brca1 and associated pathway genes), or expressing oncogenic transgenes, are frequently used as models to determine functions of novel gene products, their mechanisms of action, and for preclinical trials of new drug responses, it seemed important to continue our examination of mouse cell responses to DNA damage and DSB repair pathways. Thus, we further investigated the parallels observed in functions of Wwox and its absence in mouse DNA damage repair pathways. The implications of the new findings and the resulting new questions are discussed here.

The Murine Brca1-BRCT Tandem Domain Is Required for Binding of Wwox to Brca1
Wwox binds to the mouse Brca1-BRCT domain, independently of the Brca1 phospholigands, pAbraxas, pBrip1, and pCtIP. Murine Brca1-BRCT domain mutations (analogous to M1775R, S1655F in human, identified in familial breast cancers) abrogate not only binding of the A, B, C complexes to the BRCT domain, but also the BRCT-Wwox interaction, i.e., MEFs expressing the Brca1 S1598F mutation, are defective in HR repair and the homozygous mutant mice are highly susceptible to tumor development [40]. These findings indicate that Brca1-BRCT domain functions are severely negatively affected by single amino acid changes and that the effects include abrogation of interactions of the domain with Wwox and the protein complexes essential for DSB repair by the HR pathway. Thus, it does not seem surprising that binding of a protein such as Wwox (46 kD) to a region within this tandem repeat domain might also interfere with the BRCT domain folded structure and essential downstream functions. Therefore, we hypothesize that Wwox binding to the mouse BRCT domain delays recruitment of Brca1-BRCT associated repair proteins to DSBs by controlling the timing of A, B, C complex formation.

Wwox Loss Accelerates Brca1-CtIP and MRN Complex Formation
The Brca1-C complex has a role in end-resection. Reczek et al. have shown that ablation of Brca1-CtIP interaction did not affect pRPA foci formation after IR in murine cells [33], likely because, without binding to Brca1, the CtIP-MRN complex can localize to DSBs and promote resection through Brca1 independent Brip1-CtIP interaction [43]. Nevertheless, Brca1-CtIP interaction speeds up resection, and resection speed can be involved in repair pathway choices [35]. Wwox co-IPs with Brip1 and CtIP, and the bindings are independent of Brca1. We believe that Wwox binding to Brip1 and CtIP acts as a brake to inappropriate HR initiation by inhibiting binding of these complexes to the Brca1-BRCT domain and preventing immediate end-resection after IR. Thus, the Brip1 and CtIP binding and associated DNA resection at DSBs in Wwox-deficient cells are key events that determine the choice of HR repair immediately post-IR ( Figure 6).
Unexpectedly, Wwox deficiency dramatically increases basal levels of Brca1-C complex components that play a key role in end-resection. In Wwox KO cells, the level of MRN complex and Brca1-CtIP binding is substantially increased, even without DNA damage induction, likely due to higher protein levels of MRN/CtIP complex components. It is worth noting that, in IR exposed WT cells, the percentage of cells containing CtIP positive foci gradually increased in a time dependent manner, but, in Wwox KO cells, we see that CtIP, essential for the MRN-induced nicking step that initiates DNA resection, is already at DSB foci at 0.5 h post IR. The increased MRN complex level may preset the cells to promote end-resection immediately after DNA damage and increase the resection speed in Wwox KO cells. Thus, the cells would initially repair DSBs only through end-resection mediated pathways, HR and SSA, which are upregulated in Wwox-deficient cells [7]. Expression levels of MRN complex components are correlated with worse prognosis and chemo/radiotherapy resistance [48][49][50], as observed in Wwox negative tumors [6,7]. Our results showed elevated protein levels of CtIP and MRN complex in both Wwoxsilenced and KO MEFs, suggesting that Wwox expression level is useful for predicting therapeutic efficacy.
. Mol. Sci. 2022, 23,3729 Figure 6. Proposed model illustrating Wwox role in repair of DSBs immedia platin exposure. In cells expressing Wwox, Wwox binding to BRCT blocks B formation and BRCA1 activation; Wwox-Brip1 interaction inhibits Brip1 med cruitment to DSBs. In the absence of Wwox, both Brca1-BRCT and Brip1 protein for interaction with CtIP and MRN complex even without IR. The pre-formed is rapidly recruited to DSBs by Brip1 and initiates resection immediately, whic promotes HR/SSA repair. These repairs are mutagenic causing IR/Platinum re Unexpectedly, Wwox deficiency dramatically increases basal leve plex components that play a key role in end-resection. In Wwox KO cell complex and Brca1-CtIP binding is substantially increased, even with induction, likely due to higher protein levels of MRN/CtIP complex worth noting that, in IR exposed WT cells, the percentage of cells conta foci gradually increased in a time dependent manner, but, in Wwox KO CtIP, essential for the MRN-induced nicking step that initiates DNA re at DSB foci at 0.5 h post IR. The increased MRN complex level may promote end-resection immediately after DNA damage and increase t in Wwox KO cells. Thus, the cells would initially repair DSBs only thro mediated pathways, HR and SSA, which are upregulated in Wwox-de pression levels of MRN complex components are correlated with wo chemo/radiotherapy resistance [48][49][50], as observed in Wwox negative results showed elevated protein levels of CtIP and MRN complex in bo and KO MEFs, suggesting that Wwox expression level is useful for pre efficacy.

Wwox Absence in Mouse Cells, as in Human, Favors the Choice of HR Repair Early after DSB Induction
Wwox absence leads to very early DSB-induced DNA end-resection and enhances pRPA, Rad51, Abraxas, Brca1, and CtIP foci formation beginning at 0.5 h post IR in mouse cells and even earlier end-resection in human cancer cells [10]. The consequent early HR repair of DSBs associated with Wwox absence is significant since it enhances the subsequent resistance to commonly used DNA damaging drugs, such as IR and carboplatin, and confers a survival advantage to Wwox-deficient cells, as we have previously shown for human ovarian and lung cancer patients [6].
We have observed that Brca1 IRIF in Wwox KO cells reached a peak at an earlier time point (2 h, post-IR) than those in WT cells, and then were reduced at 4 h after IR. We propose that, when Brca1 is released from the DSBs by degradation, and new Brca1 is synthesized, NHEJ repair may begin, and normal regulation of repair could take over. Without synchronization of cells, which itself can cause DNA damage, study of this transition is difficult in cells where different repair pathways are underway in different subpopulations of cells. In accordance with this proposal, we expect that the DNA repair pathway choices are thus normalized, such that Wwox-deficient cells begin repairing remaining DSBs by all appropriate pathways, meaning that the early HR repair is aberrant but not all DSB repair is aberrant. Thus, the Wwox deficient cells, which occur in large fractions of many breast and ovarian cancers, are resistant to killing by these agents and these resistant cells survive with accumulation of some aberrantly repaired DSBs. These surviving mutant cells in a cancer patient lead to growth of cancers that will be progressive following carboplatin treatment.
How and why does absence of Wwox expression, which occurs so frequently in many types of cancers, dysregulate the HR repair pathway so dangerously, or put another way, why do cells need a Wwox 'brake' for end-resection? Without Wwox, end-resection, a critical process to determine DSB repair pathways, occurs immediately, which inhibits NHEJ repair, the dominant DSB repair pathway. We are proposing that the normal function of Wwox, in the context of DSB repair in mouse, binds to the Brca1-BRCT tandem domain, as well as to A, B, and C proteins and inhibits formation of the Brca1-BRCT complexes, delaying initiation of end-resection at DSBs, as in Figure 6, illustrating our proposed model for Wwox function in DSB repair. Wwox binding to murine Brca1-BRCT may alter the tandem BRCT domain structure such that the phospho Abraxas, Brip1, and CtIP protein complexes do not immediately bind to Brca1-BRCT protein. Wwox-Brip1 binding would thus synergistically block the Brip1 and CtIP proteins from interaction with Brca1.
We have shown that inhibiting early occurring end-resection by Chk2 inhibition successfully killed Wwox-deficient MEFs by causing synthetic lethality, but did not affect viability of WT MEFs, suggesting that IR or platinum-based therapy, combined with inhibition of Chk2 activation could be a useful strategy to target Wwox-negative, treatment resistant cancers. Although we have not yet confirmed all aspects of the mechanisms by which Wwox executes this modulatory influence to prevent premature activation of HR, we hypothesize that generation of murine cells expressing mutations or deletions in the potential Wwox-binding motifs would lead to the phenotypes observed in this study in Wwox knockout or silenced cells. cells were incubated with secondary antibodies conjugated to Alexa Fluor for 1 h in a humidified light-resistant chamber. After washing in PBS-T, coverslips were incubated in Hoechst 33342 for nuclei visualization, and mounted with Aqua-Poly/Mount medium and dried overnight. PLA staining was performed according to manufacturer's protocol (Sigma; St. Louis, MO, USA). For focus counting, images were acquired by Zeiss Axioskop 40 microscope with 40× objective lens and foci manually quantified. Representative images were acquired by confocal microscope (Olympus Spectral FV1000, Olympus; Tokyo, Japan) with 100× objective lens. Bar graphs of the data represent quantification of averages of foci numbers of three independent experiments, generated using Graph-Pad Prism 7 software.

Western Blot and Immunoprecipitation Assays
Cells were lysed in either cold 1× RIPA buffer or low salt NP40 lysis buffer (10 mM Hepes, pH 7.6, 0.25 M NaCl, 0.1% NP40, 5 mM EDTA, 10% Glycerol) supplemented with Protease Cocktail Inhibitor tablet (Roche; Basel, Switzerland). Proteins were separated on 6-12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Amersham; Amersham, UK). Membranes were blocked with 5% skim milk in TBS-T (150 mM NaCl, 250 mM Tris-HCl pH 7.4, 0.01% Tween 20) followed by overnight incubation with specific primary antisera at 4 • C. Upon washing with TBS-T, the HRP-conjugated secondary antisera were added for an hour at room temperature. After washing with TBS-T, immunoreactive proteins were detected using SuperSignal TM , West Pico PLUS Chemiluminescent Substrate (Cat# 34579, Thermo Fisher Scientific; Waltham, MA, USA). For IP, cells were harvested at 48 h after transfection, and 500 µg whole cell lysates or soluble nuclear extract were incubated with 20 µL Myc or HA-magnetic beads, overnight at 4 • C. After multiple washes, protein complexes were eluted by boiling the beads for 5 min in 15 µL of 2X reducing sample buffer, and precipitates separated on SDS-PAGE as described above.

Isolation of Soluble Nuclear Fraction
Cells were harvested in nonnuclear extraction buffer (0.5% NP-40, 10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA and protease inhibitors), incubated on ice for 15 min, centrifuged at 5000× g for 5 min, and supernatants collected for the nonnuclear fraction. Pellets were washed in nonnuclear extraction buffer and incubated in low salt NP40 lysis buffer for 30 min on ice, centrifuged at 16,000× g for 10 min, and the supernatants were collected as the soluble nuclear fraction.

DR-GFP Assay
Green fluorescence protein (GFP) reporter assays were performed using two independent MEF clones carrying the DR-GFP reporter construct in the Pim1 locus as described previously [51]. Briefly, I-SceI or empty vector were transfected by electroporation using Mirus Ingenio Solution (Mirus Bio; Madison, WI, USA), and cells were harvested 48 h after transfection. Cell pellets were resuspended in 1% FBS in PBS and transferred to 10 cm plates; after 48 h, the percentage of GFP positive cells was quantified by flow cytometry (BD TM LSR II).

Chemicals and Antisera
Carboplatin (AdipoGen, San Diego, CA, USA, AG-CR1-3591) and BML-277 (Sell-eckChem, Houston, TX, USA, Cat#S8632) were dissolved in water (10 mg/mL) and in dimethyl sulfoxide (10 mM), respectively, and stored at 20 • C. Further dilutions of each drug for the respective experiments were freshly prepared in the growth medium. Table 1 lists antisera and dilutions used for various experiments.

Single Molecule Analysis of Resection Tracks (SMART) Assay
To measure the length of resected single-strand DNA after IR, we used the SMART assay [35]. Wwox WT and KO MEFs were grown to~70% confluency and labeled with BrdU (25 µM) overnight. BrdU incubated cells were treated by IR (10 γ), trypsinized, centrifuged, and resuspended in 200 µL PBS (10 6 cells/mL) at 1 h; 4 µL of cell suspension was spotted onto the edge of a pre-marked glass slide, and allowed to air dry 5 min. The air-dried cells were mixed with 5 µL lysis buffer (0.5% SDS, 200 mM Tris-HCl, pH 7.4, 50 mM EDTA) on glass slides. After 5 min, an additional 10 µL of lysis buffer was added and slides were immediately tilted (~15 • ) to spread DNA fibers by gravitational flow. Slides were then fixed using 3:1 methanol/acetic acid for 10 min. Excess solution was drained off and fixed slides were air-dried. Slides were washed in PBS, blocked in 1% BSA in PBS and incubated with BrdU antiserum (1:100, ab6326, Abcam; Cambridge, England) for 1 h. After washing in PBS, slides were incubated in anti-Rat Alexa fluor 594 (1:500, ab150160, Abcam) and unbound antiserum removed by PBS washes. DNA fibers were stained using Hoechst 33342 and mounted with Aqua-Poly/Mount medium. Images were acquired using Zeiss Axioskop 40 microscope with 40X objective lens, and non-tangled fibers were measured using ZEN2 software. Experiments were carried out using two WT (WT3, WT7) and two KO (KO3, KO5) clones.

Colony Formation and Mutation Assays
Colony assays were performed to compare sensitivity to IR and carboplatin in Wwox KO vs. WT MEF cells with and without Chk2 silencing or inhibition. Briefly, cells were treated with different doses of carboplatin (0, 1.25, 2.5, and 5 µM) and IR (0, 0.5, 1, 2.5, 5 γ) 24 h and 2 h before seeding, respectively. Cells were washed in PBS, trypsinized, counted, and 5000 cells plated in 60 mm culture dishes in triplicate, were grown for 10-14 days and fixed, stained with crystal violet, and colony numbers counted under microscope. Percent survival is expressed as the number of colonies in the IR-treated or carboplatin-treated condition at the indicated dose of drug vs. the untreated condition. The frequency of 6-thioguanine (6-TG) resistant colonies among WT or KO MEF cells assessed the frequency of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant colonies. Cells were treated with low-dose IR (0, 0.5, 1 γ) and equally seeded (5000 cells) into 60 mm culture dishes in triplicate. Plated cells were cultured in medium containing 6-TG (Sigma, 5 µM) with medium replaced every 2-3 days. Cells were maintained at 37 • C and 5% CO 2 for 2 weeks, stained by crystal violet, and colony numbers counted by microscopic observation. Survival (%) bar graph was plotted relative to colony numbers of non-treated control cells.

Statistical Analysis
Statistical analyses were performed using an unpaired two-tailed Student's t-test to compare sets of results from independent groups, and values of p < 0.05 were considered statistically significant. Each bar graph included at least three replicates with error bars representing S.E.M and p-values calculated using Graph-Pad Prism 7 software.  Data Availability Statement: All inhouse produced materials are available upon request. The flow cytometry data for the GFP reporter assay will be publicly available at Flow Repository upon publication of the manuscript.