Oncogenic role of FEN1 in glioma patients and TMZ resistant glioma cells
To understand the expression of FEN1 in glioma cells, we used The Cancer Genome Atlas (TCGA) datasets to obtain and compare genomic data of patients with glioma and non-cancer patients. FEN1 expression was significantly overexpressed in glioma patients compared to that in non-cancer patients (Fig. 1a, Fig. S1a, Table 1). Upregulation of FEN1 expression was also found to be related to poor survival for glioma patients (Fig. 1b). A gene set enrichment analysis (GSEA) revealed significant enrichment of pathways associated with cell cycle progression, including DNA replication and the cell cycle, and homologous recombination in the glioma samples (Fig. 1c). GO analysis indicated significant enrichment for multiple pathways of FEN1 associated with cellular progression including DNA replication and MCM complex in glioma samples (Fig. 1d). These results indicated the crucial role of FEN1 in the rapid proliferation of glioma cells. The closely intertwined correlation of FEN1 and other DNA replication-related genes in glioma samples was visualized in Fig. 1e. Meanwhile, a PPI network was constructed to exhibit the interaction of FEN1-associated proteins (Fig. 1f).
To investigate the effect of high FEN1 level in glioma progression and clinical prognosis, we divided glioma samples into FEN1 high and low expression groups then investigated the FEN1-related mutational landscape. More IDH1, TP53, and ATRX mutations were observed in FEN1 low expression group, indicating a favorable prognosis that matched well with clinical diagnosis (Fig. 1g, h). Meanwhile, FEN1 high group had more PTEN and EGFR mutations and an overall higher frequency of mutation co-occurrences was uncovered, indicating an elevated mutational load (Fig. 1g-j). Increased mRNA of FEN1 and core replisome factors including MCM proteins were also observed in temozolomide (TMZ) sensitive U87MG and resistant U87MG. R cell lines by RNA sequencing (Fig. 1k). A volcano plot was graphed to show the distribution of DNA replication and repair-related genes and some significantly up-regulated members were labeled (Fig. 1l). That is, upregulated FEN1 expression results in prolonged genome stability and constitutive activation of DNA replication in TMZ resistant glioma cells.
Regarding the function of FEN1 in glioma progression, we hypothesized that a therapeutic effect can be optimized by targeting DNA replication via FEN1 inhibition and combining the effects of FEN1-mediated DNA damage signaling and clinical reagents that drive survival-related stress. In our panel of two glioma cell lines, M059K and U251 cells, we observed that FEN1 deficiency significantly and consistently reversed resistance to TMZ, cisplatin and MMS, as indicated by cell viability and survival analyses (Fig. S1b-k). Representative colony formation images indicated inhibition of the combined effects of damage reagents and FEN1 dysfunction (Fig. S1b and e). However, we did not observe significant augmentation of DNA-damage reagent cytotoxicity during FEN1 inhibition in the RPE1 non-cancer cell lines (Fig. S1f, i and k), suggesting different genetic backgrounds in the cancer and non-cancer cells and lower effects of stress-inducing agents in non-cancer somatic cells, thereby providing a promising approach to glioma clinical treatment.
FEN1 deficiency drives excessive resection of HU-arrested DNA replication forks
Replication stress induced by DNA-damaging reagents or deleterious structural changes during rapid cancer cell proliferation results in helicase-polymerase uncoupling, activating ATR through the accumulation of replication protein A (RPA)-coated ssDNA and increasing RPA chromatin binding, which triggers subsequent DNA damage signaling [34]. To investigate the role of FEN1 in glioma cells proliferation, we performed proximity ligation assays (PLAs) using specific antibodies against FEN1 and the heterotrimeric replication protein A (RPA) complex, which stabilizes ssDNA intermediates formed during DNA replication. Compared to that in undamaged cells, PLA signaling significantly increased in cells treated with hydroxyurea (HU), indicating that the association between FEN1 and RPA was enhanced when forks stalled under replication stress (Fig. 2a). The PLA signal was detected at a very low frequency with FEN1 was depleted following cell transfection with FEN1 siRNA. FEN1 deficiency led to increased native BrdU foci, also suggesting a role for FEN1 in inhibiting extensive replication stress-induced ssDNA accumulation (Fig. 2b). RPA facilitates FEN1 interaction with stalled replication forks and recruits downstream DNA repair factors and checkpoint kinases [25, 35]. Then, RPA2 is phosphorylated at serine 4 and serine 8 (S4/S8) by ATR, and phosphorylated RPA2 serves as a common marker for DSB repair processing and DNA replication stress. To test the impact of FEN1 deficiency on RPA2 phosphorylation at stalled replication forks induced by HU, we tested p-RPA (S4/S8) by immunofluorescence assays. We found that FEN1 depletion significantly resulted in increased RPA2 phosphorylation under HU-induced replication stress (Fig. S2a and b). γ-H2AX, a DSB maker, was also increased in cells transfected with FEN1 siRNA under HU treatment, shown by images of immunofluorescence labeling (Fig. 2c) and quantitation (Fig. 2d).
Persistent fork stalling induced by replication stress leads to excessive ssDNA accumulation, resulting in the insufficient levels of RPA available for ssDNA protection, an outcome called RPA exhaustion, which ultimately leads to single-strand DNA exposure, fork degradation and disabled fork restart in the absence of a DNA repair pathway [36]. Then, we investigated the performance of stalled forks with FEN1 deficiency in response to replication stress to gain further insights into the underlying mechanism critical for the increase in ssDNA accumulation in FEN1-depleted cells. First, we were interested in determining the role of FEN1 in protecting stalled replication forks from nucleolytic degradation, a function previously ascribed to several HR proteins, but not FEN1 [11, 14]. We carried out DNA fiber assays by labeling DNA with chlorodeoxyuridine (CldU) and iododeoxyuridine (IdU) and then exposed the cells to 4 mM HU for 4 h to cause fork stalling. FEN1 deficiency in M059K cells resulted in extensive shortening of nascent replication strands compared to control cells, indicating that stalled forks lead to more nuclease degradation (Fig. 2e). Continuous fork stalling and progressive shortening of nascent strands may be attributed to partial fork breakage due to fork degradation [37]. To further confirm that FEN1 is critical for protecting nascent strands from degradation, we induced fork stalling by treating cells with HU before performing IdU labeling a second time; this process has been described as a DNA dual-labeling scheme for inducing fork stalling by adding HU before adding the second halogenated nucleotide analog [38]. We found that the length of the first CldU-labeled replication strand was significantly shorter in M059K cells transfected with FEN1 siRNA (Fig. 2f), which was consistent with our previous findings showing that FEN1 can prevent excessive nucleolytic fork degradation. Collectively, these data indicate the important role of FEN1 in protecting stalled replication forks from degradation, which differs from its established function in the resection and repair of stalled replication forks.
Glioma cells deficient in FEN1 are unable to cope with replication stress impaired fork progression
Studies both in vitro and in vivo indicate that FEN1 functions in a variety of DNA processes that are mediated by several important protein–protein interactions, including those of Werner syndrome protein (WRN), one of five human RecQ helicases implicated in the maintenance of genome stability and with roles in DNA replication and repair [39]. Interacting with WRN, FEN1 has also been reported to function in restarting stalled replication forks [25]. To determine the role of the FEN1-WRN complex in the DNA replication progression of glioma cells, we next measured the fraction of stalled and active replication forks in M059K cells transfected with siFEN1, siWRN and the combination of siFEN1 and siWRN. The FEN1 and WRN protein levels are shown in Fig. S2c and indicate a decline in protein expression after siFEN1 and/or siWRN transfection. We observed excessive replication fork degradation in the siFEN1- or siWRN-transfected M059K cells (Fig. S2d). Using long-term nucleotide starvation caused by HU exposure before the IdU labeling for the second time, we found that replication progression was impaired after FEN1 or WRN depletion with a significantly increased percentage of stalled forks. After cell release from HU-induced replication stress, FEN1 or WRN depletion resulted in a more than threefold increase in the number of stalled replication forks (Fig. S2e). FEN1 or WRN depletion also resulted in a decrease the number of ongoing replication forks of more than 20% after cell release from stress (Fig. S2f). FEN1 and WRN double depletion did not show additive effects on the progression of stalled forks, confirming their common function in the same fork protection pathway. Correlation analysis of 173 glioma patient samples (r = 0.52, p < 0.001) showed that FEN1 was positively correlated with WRN expression (Fig. S2g). Collectively, our data indicate that FEN1 is required for restarting replication forks stalled due to stress and for regularly maintaining cell cycle progression.
FEN1 deficiency resulted in failure of RAD51-BRCA1 assembling and increased reversed fork degradation
Our and other studies have reported that fork remodeling by replication fork reversal allows DNA synthesis to pause and resume once the block is removed, serving as a mechanism for cells to maintain genomic stability upon replication stress [10, 12]. Mechanisms of fork protection that contribute to replication fork stability also endow cancer cells with chemo-resistance [13]. The key factors in homologous recombination (HR), BRCA1 and BRCA2, are important for stabilizing reversed forks and preventing extensive nuclease resection, as regressed arms act as entry points for unsolicited MRE11 degradation in BRCA-deficient cells [40]. Then, we studied the mechanism of FEN1 in protecting stalled replication fork degradation in response to replication stress. We labeled FEN1, BRCA1 and RAD51 in glioma mouse samples and observed decreased BRCA1 and RAD51 expression upon treatment with the specific FEN1 inhibitor sc13 [41], confirming the regulatory role of FEN1 on BRCA1 and RAD51 in glioma cells (Fig. 3a and b). Consistent with these results, there was a significant positive correlation between FEN1 and BRCA1 expression (r = 0.65, p < 0.001) and RAD51 expression (r = 0.56, p < 0.001) in the TCGA data (Fig. 3c and d). GSEA revealed significant enrichment of pathways including DNA replication, cell cycle, and homologous recombination with BRCA1 and RAD51 in the glioma samples (Fig. S3a and b). Elevated BRCA1 and RAD51 level were also observed in glioma patient samples compared with normal samples (Fig. S3c). As FEN1 depletion has been reported to impair damaged replication fork repair processing by reducing BRCA1/RAD51 function, we examined foci formation and found significantly decreased BRCA1 and RAD51 foci formation in M059K cells transfected with FEN1 siRNA compared to un-transfected cells, indicating decreased BRCA1/RAD51 recruitment to stalled forks (Fig. 3e) and markedly inhibited BRCA1-RAD51 assembly following HU treatment and observed PLA protein–protein in situ interactions in FEN1-deficient cells (Fig. 3f). Therefore, from these data, we derived a BRCA1 and RAD51 co-expression signature that demonstrated high concordance with the FEN1 signature in terms of the involved pathways and regulation by FEN1 inhibition. Therefore, FEN1 is a key regulator of BRCA1 and RAD51 protein levels and function in glioma cells (Fig. 3a-f).
We also monitored DNA strand degradation with a DNA fiber assay. As shown in Fig. 3g and Fig. S3d, HU-treated FEN1- or BRCA1-deficient cells displayed substantial degradation of nascent DNA strands, while levels of replication in wild-type cells were restored and MRE11 expression was depleted, indicating that FEN1 protect stalled replication forks from excessive MRE11 resection by stabilizing BRCA1 at stalled replication forks. BRCA1 was also critical for promoting RAD51 loading upon replication stress. Similar replication fork degradation with FEN1 depletion resulted in BRCA1 deficiency, and fork breakage was also observed in cells transfected with RAD51 siRNA (Fig. 3h). In addition, RAD51 has also been reported to be required for the accumulation of reversed forks, protecting the stalled forks in a BRCA2-independent manner in response to replication blocks [42]. In contrast to a previous report suggesting that knockdown of RAD51 can fully restore fork degradation in the absence of CTIP [43], we found that cells with RAD51 depleted displayed fork degradation to the same extent as when FEN1 is knocked down, and double depletion of FEN1 and RAD51 did not trigger fork degradation, suggesting that FEN1 and RAD51 are involved the same regulatory pathway and that they show functional variations in glioma cells under certain conditions (Fig. 3i). Protein levels were determined by in cells with siRNA transfection by western blotting. Similarly, decreases in RAD51 expression have been shown to reduce glioma cells capacity for DNA repair and increased glioma cells sensitization to radiotherapy [44]. FEN1 depletion is a promising approach to glioma therapy by reducing HR-mediated DSB repair capacity.
As extended nascent DNA strand degradation and unstable reversed forks were observed in HU-treated BRCA2-deficient cells, we monitored the influence of FEN1 depletion on BRCA2 recruitment to stalled forks as well as its function on replication forks upon HU stress. The phenotype of the BRCA2-deficient cells with excessive DNA strand degradation was similar to that of BRCA1-deficient cells, while MRE11 depletion restored the full replication progression in BRCA2- or FEN1-deficient cells (Fig. S3e). We also observed decreased BRCA2 foci formation with BRCA2 depletion, similar to that observed in FEN1 deficiency (Fig. S3f). Protein levels were determined in cells with siRNA transfection by western blotting (Fig. S3g). The working model generated to study the role of FEN1-BRCA1/2-RAD51 in protecting replication forks against MRE11 degradation (Fig. S3h). These findings indicate that FEN1 functions in stalled replication fork protection in a BRCA-dependent manner following DNA replication stress induced by HU treatment.
Fork degradation is replication fork reversal dependent in FEN1 deficient cells
Replication fork reversal is a phenomenon of stalled replication fork remodeling that allows temporary DNA synthesis and protects stalled fork integrity upon DNA replication stress [10]. By reannealing the nascent DNA strands to form a fourth regressed arm, stalled replication forks are remodeled into a “chicken foot” structure. DNA-damaging agents, protein-DNA complexes, or nucleotide depletion induced by hydroxyurea (HU) treatment have been shown to cause replication stress resulting in ssDNA formation and threaten genome stability [7, 8]. Fork reversal has been shown to prevent ssDNA accumulation, promote template switching and error-free lesion bypass and restore replication progression [45, 46]. Thus, fork reversal is considered a protection mechanism that resolves stalled replication forks and maintains genomic stability in response to replication stress. SMARCAL1, ZRANB3, and HLTF, three members of the SNF2 family of DNA translocases, are thought to catalyze fork reversal in response to HU-induced nucleotide depletion and replication fork stalling [47,48,49]. The regressed arm of reversed forks has been shown to serve as an initiation point for uncontrolled MRE11-dependent degradation in BRCA-deficient cells [40, 50]. Thus, to investigate whether FEN1 elicits its fork-protection function before or after fork remodeling, we measured the IdU: CldU ratio in DNA fibers of M059K cells transfected with FEN1 siRNA in combination with either a SMARCAL1, ZRANB3, or HLTF siRNA. As shown in Fig. S4a, depletion of each individual DNA translocase abolished fork degradation in the FEN1-deficient cells. These data suggest that fork reversal is a prerequisite for triggering extensive nascent strand degradation in FEN1-deficient cells upon HU-induced replication stress.
Moreover, DNA2 was recently reported to extensively resect the regressed arms in CTIP-deficient cells [43]. Therefore, we examined whether DNA2 is critical for nascent DNA strand degradation in FEN1-deficient cells. However, in contrast to the results obtained upon depletion of MRE11expression, co-depletion of DNA2 and FEN1 expression did not attenuate fork degradation following HU treatment, indicating that depletion of DNA2 did not restore fork stability in the FEN1-deficient cells (Fig. S4b). Protein expression was detected with the indicated antibodies (Fig. S4c and d). Collectively, these data indicate that FEN1 protects reversed replication forks from MRE11-nucleolytic attack, depending on BRCA promotion of RAD51 loading and stabilization of nucleofilaments, not by DNA2 cleavage.
Tumor evolution drives glioma cells reliant on FEN1-dependent proliferation with DNA-PKcs Deficiency
Cells utilize multiple mechanisms to maintain genome stability in response to replication stress-induced DNA damage, particularly DSB damage in cancer cells carrying the capacity for high replication progression to support their proliferation. The loss of single genes in DSB repair is not lethal because cancer cells rely on alternative DNA repair pathways. Therefore, high capacity of an effective and complementary DSB repair system contributes greatly to chemo- and radio-resistance. We next sought to determine whether FEN1 and DNA-PKcs co-depletion can block different signaling-mediated fork integrity safeguards in response to HU-induced fork stalling and DSB formation, leading to synthetic effects. Firstly, we observed significantly elevated amounts of FEN1 were recruited to replisomes as indicated by PLA analysis between FEN1 and replisomes components MCM2 and MCM5 in M059J cells (Fig. 4a and c). This finding corroborates observations in glioma cells with DNA-PKcs deficiency and in M059K cells transfected with DNA-PKcs siRNA (Fig. 4b and d) and indicates tumor evolution shifts caused by enhanced interaction between FEN1 and replisomes in cases of DNA-PKcs deficiency and FEN1 contributes to structured replisomes during cell circle. The co-immunoprecipitation assay confirmed these interactions which confirmed the enhanced interaction between FEN1 and replisomes in DNA-PKcs deficient cells (Fig. 4e). The association between FEN1 and MCM proteins was positive in control cells and increased in HU-treated cells, in agreement with the PLA assay results. These data demonstrate the shift function of FEN1 on DNA replication machine and the enhanced association of FEN1 with replisomes and alternative functions in the progression of glioma cells with DNA-PKcs dysfunction in response to replication stress.
FEN1 deficiency triggers impaired fork progression and fork degradation in DNA-PKcs deficient glioma cells
We next carried out a DNA fiber spreading assay to examine the co-effect of FEN1 and DNA-PKcs deficiency on stalled replication forks. We observed significant fork degradation in replication-induced M059J cells upon HU treatment and triggered DNA strand resection combined upon FEN1 depletion (Fig. 4f). Then, we assayed the effect of dual depletion of FEN1 and DNA-PKcs on replication in M059K and M059J cells exposed to HU before the second DNA strand was IdU-labeled. An excessively shortened CldU-labeled strand length was observed in the M059J cells with both FEN1 and DNA-PKcs depleted (Fig. 4g). Similar phenotypes were observed in different glioma cell lines, namely, U251 and U87MG cells that were transfected with siFEN1 and siDNA-PKcs (Fig. 4h, i). FEN1 depletion resulted in an increased stalled fork frequency and a further decrease in fork progression in the M059J cells transfected with FEN1 siRNA (Fig. 4j, k).These findings indicate the synthetic effect of both FEN1 and DNA-PKcs deficiency on extensive stalled fork degradation in response to HU-induced replication stress. Protein expression significantly decreased after siFEN1 and siDNA-PKcs treatment, as determined by western blot assay (Fig. S5a-b).
To confirm this conclusion, we also analyzed fork degradation upon effective FEN1 and DNA-PKcs depletion by treating two additional glioma cell lines, U251 and U87MG cells, with the FEN1 inhibitor sc-13 [41] and the DNA-PKcs inhibitor NU-7441[51], which can be used both in vitro and in vivo. Compared to the effects on cells treated with either sc-13 or NU-7441 individually, FEN1 and DNA-PKcs dysfunction induced by treatment both inhibitors combined dramatically triggered fork degradation following HU stress induction (Fig. S5c). The same results were observed in M059J cells treated with sc-13, and fork degradation was blocked by the MRE11-specific inhibitor mirin (Fig. S5d). Triggered stalled fork frequency and a further decrease in fork progression was observed in multiple glioma cells with both FEN1 and DNA-PKcs dysfunction after specific inhibitor treatment (Fig. S5e-h). Collectively, these data imply the synthetic effect of FEN1 and DNA-PKcs depletion that leads to extensive stalled fork degradation, impaired fork restarting and additive genetic interactions in the maintenance of stalled replication fork integrity in response to HU-induced replication stress.
Combined FEN1 and DNA-PKcs deficiency promotes replication-stress-induced DSB formation and genome instability
Prolonged replication fork stalling and failure to effectively complete replication during the S phase lead to fork collapse or breakage, followed by replication-coupled DSB damage and fork instability [52]. Therefore, we next examined the synergy between FEN1 and DNA-PKcs in response to replication stress and investigated the further functional interplay between FEN1 and DNA-PKcs on glioma cell growth. Co-inhibition of FEN1 and DNA-PKcs in M059K cells with sc-13 and NU-7441 treatment led to a significantly higher tail moment in a comet assay than that in an assay of cells treated with sc-13 or NU-7441 separately, implying attenuated DNA repair capacity of different DSB repair mechanisms in cells with deficiency in both FEN1 and DNA-PKcs (Fig. 5a and e). Moreover, γ-H2AX foci formation is widely used as a DSB damage marker, and using immunofluorescence staining, we found only mild γ-H2AX foci formation in cells with either FEN1 or DNA-PKcs deficiency. However, we observed an extended accumulation of γ-H2AX foci in FEN1/DNA-PKcs-co-deficient M059K cells (Fig. 5b and f). A similar phenotype was observed with respect to focus formation of 53BP1, another DSB marker, with results showing a synthetic effect increase in FEN1/DNA-PKcs-deficient cells (Fig. 5c and f). These findings confirmed the synthetic effect of replication fork breakage and the ultimate result of DSB accumulation upon both FEN1 and DNA-PKcs deficiency. More importantly, we observed that the combined dysfunction of FEN1 and DNA-PKcs gave rise to a striking increase in chromosomal aberrations and micronuclei formation compared to the effect of only FEN1 or DNA-PKcs depletion, suggesting extensive chromosomal instability (Fig. 5h and i). These results provide strong evidence to support the theory of the complementary interaction of FEN1 and DNA-PKcs in counteracting replication stress-induced accumulation of damaged DNA and genomic instability.
Disruption of FEN1 and DNA-PKcs exacerbates impaired cellular progression and growth reduction
We next monitored the effect of FEN1/DNA-PKcs co-inhibition on glioma cells cycle progression and cell growth and observed that combined depletion of FEN1 and DNA-PKcs resulted in a reduction in EdU incorporation compared with cells depleted of either protein alone (Fig. 6a and b), suggesting that the synthetic interaction between FEN1 and DNA-PKcs counteracts DNA replication upon endogenous stress-induced impaired cell cycle progression by inhibiting DNA synthesis in the S phase. The synthetic toxicity of FEN1 and DNA-PKcs deficiency on cell growth was also observed as a reduction in the clonogenic survival fraction and short-term (four-day exposure) viability of U251 cells treated with sc-13 and NU-7441 (Fig. 6c-e). The same excessive survival inhibition was obtained in DNA-PKcs-deficient M059J cells after FEN1-specific small-molecule inhibitor treatment, compared with DNA-PKcs-proficient M059K cells (Fig. S6a-c). Strong invasion and migration are distinctive characteristics of glioma cells; therefore, we next investigated the influence of FEN1 and DNA-PKcs inhibition on the invasion and migration ability. As expected, dramatically decreased invasion and migration abilities were observed for cells with both FEN1 and DNA-PKcs depleted (Fig. 6f-h). Similar findings of excessive inhibition of invasion and migration were observed in M059J cells with combined FEN1 and DNA-PKcs depletion (Fig. S6d and e). Triggered toxicity was found in M059J cells treated with increasing concentrations of the inhibitor sc-13 (Fig. 6i-k). A similar influence of sc-13 combined with another DNA-PKcs inhibitor, vx-984, was observed in multiple glioma cells, but the extent of the effects was different in other cancer cell lines, such as A549 and Huh-7 cells, reflecting promising targeting of FEN1/DNA-PKcs in tumor therapy (Fig. 6l). Collectively, these findings consistently support a scenario in which deficiency of FEN1 and DNA-PKcs causes cell growth inhibition and aggressive decline in the migration and invasion of glioma and other tumor cells.
In vivo FEN1/DNA-PKcs synthetic lethality
We next assessed whether combination treatment with FEN1 and the DNA-PKcs inhibitors sc-13 and NU-7441 can inhibit glioma tumor establishment in vivo. First, we generated cohorts of null mice with xenograft tumors derived from luciferase-labeled U87MG cells. Once tumors were established, the mice were treated with either vehicle, sc-13, NU-7441 or both inhibitors for 4 weeks (Fig. 7a). The signal in the region of interest (ROI) was measured at different time points after drug treatment for up to 4 weeks. The combination of sc-13 and NU-7441 treatment significantly inhibited tumor growth compared to vehicle or sc-13 or NU-7441 treatment only. The tumor volume with sc-13 and NU-7441 treatment was dramatically lower than that of the other control groups (Fig. 7b and c). A dramatically increased surviving fraction was observed in the group of mice treated with sc-13 and NU-7441 (Fig. 7d). Sectioned brain tissue was assessed with H&E staining to determine the tumor structure accurately (Fig. 7e). As observed through immunohistochemistry labeling, significantly reduced Ki67 levels confirmed the inhibition by sc-13 and the synthetic effect of NU-7441 on glioma cells proliferation. Elevated levels of TUNEL also indicated increased tumor cell apoptosis. The levels of BRCA1 and RAD51 were significantly decreased upon sc-13 treatment, and PARP1 expression was largely reduced upon NU-7441 treatment, results in line with the findings of in vitro experiments. The superimposed deduction of BRCA1, RAD51 and PARP1 with both sc-13 and NU-7441 treatment indicated the two essential pathways critical for tumor cell replication and genome stability in response to replication stress (Fig. 7e-j).
Collectively, these in vivo experiments confirmed the synthetic lethality between FEN1 and DNA-PKcs in glioma cells.