GSK3β Inhibition Synergizes with PARP Inhibitors through the Induction of Homologous Recombination Deciency in Colorectal Cancer

Background: Monotherapy with poly ADP-ribose polymerase (PARP) inhibitors results in limited objective response rate ( ≤ 60% in most cases) in patients with homologous recombination repair (HRR)-decient cancer, which suggests a high rate of resistance in this subset of patients to PARP inhibitors (PARPi). To overcome resistance to PARPi and to broaden their clinical use, we performed high-throughput screening of 99 anticancer drugs in combination with PARPi to identify potential therapeutic combinations. Methods: The effects of PARPi combined with glycogen synthase kinase 3 (GSK3) inhibitors were investigated in vitro with respect to cell viability, cell cycle and apoptosis. The synergy was assessed by calculation of the combination index (CI). GSK3α null and GSK3β null cells were generated using CRISPR/Cas9 technique. The underlying mechanism was examined by western blotting, ow cytometry, qRT-PCR and uorescence microscopy. This combination was also evaluated in the mouse xenograft model; tumor growth and tumor lysates were analyzed, and the immunohistochemistry assay was performed. All data are presented as mean ± SD. Comparison between two groups was performed with the Student’s t-test. Result: The data showed that ~25% of oncological drugs and kinase inhibitors that were evaluated displayed synergy with PARPi in HCT-15 cells. Among the tested agents, GSK3 inhibitors (GSK3i) exhibited the strongest synergistic effect with PARPi. Moreover, the synergistic antitumor effect of GSK3 and PARP inhibition was conrmed in a panel of colorectal cancer (CRC) cell lines with diverse genetic backgrounds. Additionally, inhibition or genetic depletion of GSK3β was found to impair HRR of DNA and reduce the mRNA and protein level of BRCA1. Finally, we demonstrated that inhibition or depletion of GSK3β could enhance the in vivo sensitivity to simmiparib without toxicity. Conclusion: Our results provide a mechanistic understanding of combination of PARP and GSK3 inhibition, and support the clinical development of this combination therapy for CRC patients. therapy strategies were investigated to improve the anticancer ecacy in BRCA-mutated cells with primary PARPi resistance. We rst performed a preliminary screening of 99 anticancer drugs in combination with the PARPi, olaparib and niraparib, in HCC1937 or HCT-15 cells. The results revealed that inhibition of PARP partly affected the cellular sensitivity to a panel of oncological drugs and kinase inhibitors. Among these agents, glycogen synthase kinase 3 (GSK3) inhibitors (GSK3i) exhibited the best synergistic effect with PARPi in BRCA2-decient HCT-15 cells. Moreover, the data showed that the PARPi, simmiparib, acted synergistically with the GSK3i, CHIR99021 HCl and LY2090314, in a panel of BRCA-procient CRC cells. These results indicated that combination of GSK3i and PARPi may serve as a new therapeutic strategy for CRC patients.

Currently, the combination of olaparib and bevacizumab has been approved for patients with advanced ovarian cancer [7].
PARPi are thought to trap the PARP1/2 enzymes at site of DNA damage, leading to replication-induced DNA damage that requires BRCA1/2-dependent homologous recombination repair (HRR) [8]. Therefore, DNA-damaging agents and molecular inhibitors that target DNA damage response pathways, such as ATR and Chk1, are expected to enhance the antitumor effect of PARPi [9,10]. PARPi in combination with other targeted therapies that are capable of disrupting HRR have also shown promising results in preclinical studies [6]. However, clinical studies showed that PARPi in combination with cytotoxic chemotherapies (such as topotecan, cisplatin, gemcitabine and temozolomide) had limited clinical e cacy and high toxicity [11][12][13][14]. Therefore, new combination strategies are needed to improve the e cacy and alleviate the toxicity of combination therapy.
Clinical studies showed that more than 40% of BRCA1/2-de cient patients failed to respond to PARPi, which meant a high rate of de novo resistance to PARP inhibition even among in BRCA-mutated tumors [4,5]. Previous data revealed that PARPi including olaparib, niraparib and simmiparib induced mild synthetic lethality in human breast cancer, HCC1937 (BRCA1-de cient), and colorectal cancers (CRC), HCT-15 (BRCA2-de cient), cells in vitro, and the antitumor activity was limited in mouse xenograft models [15][16][17]. Thus, HCC1937 and HCT-15 cells serve as model cell lines for de novo resistance to PARP inhibition. Specially, majority of studies focused on a speci c drug and PARP inhibition-induced alterations of its e cacy in BRCA-pro cient cancer cells. As such, relatively little is known about the BRCA1/2 de ciency on PARPi-based combination and how PARPi alters the e cacy of a broad spectrum of drugs.
In this study, new combinatorial therapy strategies were investigated to improve the anticancer e cacy in BRCA-mutated cells with primary PARPi resistance. We rst performed a preliminary screening of 99 anticancer drugs in combination with the PARPi, olaparib and niraparib, in HCC1937 or HCT-15 cells. The results revealed that inhibition of PARP partly affected the cellular sensitivity to a panel of oncological drugs and kinase inhibitors. Among these agents, glycogen synthase kinase 3 (GSK3) inhibitors (GSK3i) exhibited the best synergistic effect with PARPi in BRCA2-de cient HCT-15 cells. Moreover, the data showed that the PARPi, simmiparib, acted synergistically with the GSK3i, CHIR99021 HCl and LY2090314, in a panel of BRCA-pro cient CRC cells. These results indicated that combination of GSK3i and PARPi may serve as a new therapeutic strategy for CRC patients.

Cell lines
Human HCC1937, HCT-15, RKO, HCT-116, and HT-29 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). SW480 and SW620 cell lines were obtained from the Cell Bank of the Chinese Academy of Sciences Type Culture Collection (Shanghai, China). DR-U2OS cells were gifted by Ming Huang (Shanghai Institute of Materia Medica). Cells were cultured according to the supplier's instructions and authenticated by short tandem repeat (STR) analysis performed by Genesky.
Screening of drug combinations BRCA1-de cient HCC1937 and BRCA2-de cient HCT-15 cells were used for screening the drug combinations. Prior to screening, olaparib and niraparib were arrayed in 96-well plates and serially diluted 2-fold to obtain a concentration that was 20% of inhibition rate (IR) of olaparib and niraparib.
Sulforhodamine B (SRB) assay was used to analyze cytotoxicity. For combination screening, cells were plated and then treated with the various agents at three concentrations, covering a 100-fold concentration range, with or without a xed concentration of olaparib or niraparib (~20% IR). The IRs of the various agents, PARPi and combinations were de ned as IR1, IR2, and IR3, respectively. The combination effect was calculated using the IR values and equalled to IR3-IR2-IR1, which was de ned by the color intensity (green [0% inhibition] to red [100% inhibition]).
Cytotoxicity assays and combination analysis Cells were treated with the indicated drug combinations and the IR on cell proliferation was determined using SRB assays as described previously [19].
individual genes in DNA double-strand breaks (DSBs) repair, the cells were treated with the indicated agents or transfected with siRNA for 24 h. Then, the cells were transfected with a plasmid expressing I-SceI (pCBASce) for 48 h. GFP-positive cells were quanti ed using ow cytometry.

Western blotting
Standard western blotting protocol was used to measure the cellular level of the indicated proteins, as described previously [17].

In vivo anticancer activity experiments
Female nu/nu athymic BALB/cA mice (aged 5-6 weeks) were obtained from GemPharmatech (Jiangsu, China). All studies were conducted in compliance with the Institutional Animal Care and Use Committee guidelines of the Shanghai Institute of Materia Medica (Shanghai, China).
HCT-15, RKO, and HCT-15 KO xenografts were established by inoculating 5 × 10 6 cells subcutaneously in the nude mice. When the xenografts reached a volume of 60-100 mm 3 , the mice were randomized into control and treatment groups as indicated. Simmiparib and LY2090314 alone or in a combination were injected every other day for the indicated period. Tumor growth was monitored by the measuring the tumor size using calipers every other day and the tumor volume was calculated using the formula (length × width 2 )/2. Tumor tissues were collected 2 h after nal dosing for immunoblotting or immunohistochemical staining. Images of immunohistochemical staining were captured using a NanoZoomer S210 (Hamamatsu, Japan) and processed using the NDP.scan.3.2.15 software.

Statistical analyzes
All data are presented as mean ± standard deviation (SD). Comparison between two groups was performed using the Student's t-test. p < 0.05 was considered to be statistically signi cant.

Results
Drug combination screen identi es GSK3i as acting synergistically with PARPi To explore whether small-molecule inhibitors can sensitize cancer cells to PARPi, we performed a drug combination screen in BRCA1-de cient breast cancer cell line of HCC1937 and BRCA2-de cient CRC cell line of HCT-15, which express mutant-type BRCA1 or BRCA2 protein but modestly respond to PARPi. FDAapproved PARPi (Olaparib and Niraparib) and 99 well-characterized anticancer drugs targeting fty classes of proteins belonging to indicated different kind of signaling pathway were chosen for initial screen (Table S1). Strikingly, a strong synergistic effect of GSK3i (CHIR99021 HCl and LY2090314) and PARPi (Olaparib and Niraparib) was observed in HCT-15 cells ( Figure 1A). Unsurprisingly, ATR inhibitors and CHEK1 inhibitors showed synergistic effects with PARPi (Olaparib and Niraparib) in HCC1937 and HCT-15 cells, which had been reported that both ATR and CHEK1 inhibitors increased the sensitivity to PARPi in a BRCA1-independent way [9,10]. Moreover, prior studies have demonstrated that inhibitor of BET, CDK1, HDAC, Protease, PI3K, and VEGFR could all decrease BRCA1 and other HRR factors at the protein level, thereby increasing the sensitivity of the cancer cell lines to PARP inhibition [24][25][26][27][28][29]. Consistent with the above conclusion, we found that these inhibitors displayed a synergistic effect with Olaparib and Niraparib in HCT-15 cells ( Figure 1A). As reported [30][31][32][33], we also observed that Olaparib and Niraparib showed a synergistic effect in combination with inhibitors of DNMT, DNA-PK, mTOR, and HDM in HCT-15 cells ( Figure 1A).
To further con rm the accuracy of our screening results, we validated the above results in the combination of Olaparib and CDK1 inhibitor (RO-3306) or ATR inhibitor (VE821) using the CalcuSyn model. Both combinations (i.e., olaparib + RO-3306 and olaparib + VE-821) caused obvious synergistic effects (CI < 0.7) in BRCA2-de cient HCT-15 cells, while only the olaparib and VE-821 combination produced synergistic effect (CI < 0.6) in the BRCA1-de cient HCC1937 cells ( Figure 1B and 1C). These data were consistent with the observation shown in Figure 1A.

GSK3 inhibition sensitizes BRCA-pro cient CRC cells to PARPi
We next sought to validate the observed interactions between GSK3 activity and PARPi. To further investigate the effect of GSK3 activity on cellular response to PARPi, two speci c GSK3i, LY2090314 (LY) and CHIR99021 HCl (CHIR), were used in combination with ve PARPi, including olaparib, niraparib, rucaparib, talazoparib and simmiparib. To exclude the synergistic effects were simply due to cell cycle arrest, we chose the concentrations of GSK3i (CHIR ≤ 10 μM; LY ≤ 5 μM) that had no discernible effect on cell proliferation ( Figure S1A) and cell cycle phasing. Cells were treated with PARPi at eight concentrations, with or without LY2090314 or CHIR99021 HCl. The data showed that GSK3 inhibition strongly synergized with simmiparib (SP), talazoparib (TP), olaparib (OP), rucaparib (RP) and niraparib (NP) in HCT-15 cells (Figure 2A). The synergistic effect decreased in the order of simmiparib (sensitive fold: up to ~4463-fold), talazoparib (~185-fold), olaparib (~10-fold), niraparib (~4-fold) and rucaparib (~3-fold) when combined with LY2090314. Thus, simmiparib, a potent and selective PARP inhibitor currently in phase I clinical trials in China, was the most strongly perturbed following GSK3 inhibition (No. CTR20160475). Moreover, the presence of GSK3i led to a decrease IC 50 of simmiparib in a concentrationdependent manner in HCT-15 cells ( Figure 2B and Figure S1B). In line with the synergistic effects between simmiparib and GSK3i, we observed enhanced G2/M arrest and apoptotic cell death induced by simmiparib when combined with LY2090314 ( Figure 2C-E) or CHIR99021 HCl (Figure S1C-E). The protein levels of cleaved PARP1 (p85) and cleaved-Caspase 3 increased accordingly ( Figure 2F and Figure S1F). The results indicated that simmiparib and GSK3i combination treatment signi cantly suppressed tumor cell growth, caused cells to accumulate in G2/M of the cell cycle and induced remarkably apoptotic response.
To determine whether these synergies extend across other tumor cells, we used additional BRCApro cient CRC cell lines (RKO, HCT-116, SW480, SW620, and HT-29). The data showed that GSK3 inhibition strongly synergized with simmiparib in all the BRCA-pro cient CRC cells (CI < 0.6), as well as HCT-15 cells ( Figure 2G and Figure S1G). Consistently, no combination activity (CI > 1) was observed in BRCA1-de cient HCC1937 cell lines ( Figure S1H). This nding suggested a broader bene t of PARPi combined with GSK3i in BRCA-pro cient CRC cells.
GSK3β depletion selectively sensitizes cancer cells to PARP and topoisomerase (Top) I inhibitors There are two highly homologous forms of GSK3 in human, GSK3α and GSK3β, that have different tissue-speci c functions and substrates [34,35]. As GSK3i (LY2090314 and CHIR99021 HCl) block both GSK3α and GSK3β activity, we next generated GSK3α null and GSK3β null cells lines using CRISPR/Cas9 technique in HCT-15 and RKO cells, respectively ( Figure 3A and 3B). Relative to the parental cells, the GSK3β KO cells (#KO1 and #KO2) displayed up to 60-fold increased sensitivity to the PARPi, simmiparib ( Figure 3C and 3D). However, GSK3α depletion did not affect the cellular sensitivity to PARPi ( Figure 3E).
These results indicated that depletion of GSK3β selectively sensitized cancer cells to PARPi.

GSK3β is required for the HRR of DSBs
Although PARPi and Top I inhibitor cause different forms of DNA lesions, both agents are known to selectively kill proliferating cancer cells by causing replication-dependent DSBs [36,37]. For this reason, we compared the occurrence of drug-induced DSBs in GSK3β KO and parental cells, using γH2AX as a marker. Higher level of γH2AX protein accumulated in GSK3β KO cells compared to the parental cells ( Figure 4A and Figure S3A). This result was further supported by the enhanced γH2AX protein level in cells treated with a combination PARPi and GSK3i ( Figure 4B and Figure S3B); and the observation was recapitulated using an immuno uorescence assay to stain nuclear γH2AX foci ( Figure 4C and Figure  S3C). However, the level of DSBs was similarly induced in GSK3α null cells and parental cells ( Figure  S3D). These results indicated that a defect in DSBs repair was caused by the knockout of GSK3β, but not GSK3α.
Replication-dependent DSBs lesions are known to be predominantly repaired by HR, a repair process requiring homologous DNA sequence as a template. To test whether GSK3β inhibition and knockdown cells were defective in HRR, we chose a well-characterized reporter assay using the DR-U2OS, a human osteosarcoma cell line with chromosomally integrated HR reporter gene containing an I-SceI recognition sequence [23]. In this cell line, HRR using a direct repeat within the reporter cassette as a template results in an intact GFP gene, which can be detected by ow cytometry. The data showed that GSK3β knockdown using two independent siRNAs remarkably decreased the HR e ciency triggered by I-SceI ( Figure 4D). Consistently, the GSK3i, CHIR99021 HCl and LY2090314, signi cantly reduced the capacity of HRR, in which ATR inhibitor, VE821, was used as a positive control ( Figure 4E). However, GSK3α silencing had no impact on HR e ciency ( Figure S3E). Additionally, we observed impaired RAD51 foci formation in GSK3β KO cells or GSK3i-treated cells which further strengthened the de ciency in HRR ( Figure 4F and Figure S3F). Together, these data identi ed a previously unappreciated role of GSK3β in HRR, which echoed our ndings that GSK3β inhibition and depletion affected cell sensitivity to PARP and Top I inhibitors.

GSK3β depletion represses the expression of BRCA1
To understand how GSK3β is involved in HRR, we analyzed the protein level of the key factors involved in the HR pathways using western blotting. GSK3β KO cells showed a marked reduction in BRCA1 protein levels, whereas the levels of Mre11, CtIP, RPA32 and RAD52 were not affected ( Figure 5A and Figure S4A). Similarly, inactivation of GSK3β by CHIR99021 HCl and LY2090314 treatment led to a marked decrease in BRCA1 protein level in a concentration-and time-dependent manner ( Figure 5B and 5C; Figure S4B and S4C). Furthermore, we found that GSK3β depletion and inhibition reduced RAD51 protein level in HCT-15 cells but not in RKO cells (Figure5A and 5B, Figure S4A and S4B). Therefore, we assessed the effect of LY2090314 on BRCA1 and RAD51 protein levels in other CRC cells (HCT116, HT29, SW480 and SW620). LY2090314 modestly decreased RAD51 level in HT-29 cells, while it consistently decreased BRCA1 protein in all the lines assessed ( Figure S4D). We thus focused on BRCA1 as a likely mediator of the GSK3i effect. We transfected WT-GSK3β (WT) or a kinase-inactive mutant GSK3β Y216F (Y216F) cDNA into HCT-15 KO cells, and obtained the corresponding variants that expressed the WT or Y216F GSK3β proteins. As expected, reconstitution with WT-GSK3β, but not Y216F-GSK3β, partially restored the BRCA1 protein level ( Figure 5D), suggesting that GSK3β enzymatic activity was required to retain protein. Ectopically expressed FLAG-GSK3β also resulted in an increase in BRCA1 protein in the parental HCT-15 cells, which further suggested a strong association between GSK3β and BRCA1 ( Figure 5E). The reduction in BRCA1 protein level appeared to be a result of transcriptional repression, as RT-PCR revealed that the GSK3β KO cells had reduced BRCA1 mRNA expression ( Figure 5F and Figure S4E). In addition, cells treated with GSK3i (CHIR99021 HCl and LY2090314) showed a reduced mRNA expression of BRCA1 in a time-and concentration-dependent manner ( Figure 5G and S4F). However, BRCA1 protein levels were not affected by MG132 treatment in GSK3β KO or GSK3i-treated cells ( Figure 5H). Collectively, these data implied that GSK3β may repress BRCA1 transcription and protein expression in an enzymedependent manner.

PARPi and GSK3β inhibition are synergistic in vivo
Our data thus far indicated that GSK3 inhibition strongly synergizes with PARPi in BRCA2-de cient and BRCA1/2-pro cient cancer cells in vitro. We further validated this therapeutic potential using xenograft mice models. BRCA2-de cient HCT-15 cells and BRCA-pro cient RKO cells were subcutaneously injected into nude mice, and once tumor volume reached ~70 mm 3 , either simmiparib or LY2090314, alone or in combination, was injected every other day for 14 d. Notably, the combination of these two agents signi cantly inhibited the growth of the tumor in the HCT-15 and RKO xenograft mouse model, although the tumor growth in the single agent groups was not affected following simmiparib or LY2090314 treatments ( Figure 6A and 6B). Consistently, the tumor burden was signi cantly reduced as measured by the weight of dissected tumors ( Figure 6C and 6D). The increased response to the combination treatment was associated with increased number of DSBs lesions (as indicated by γH2AX levels), as well as increased the levels of cleaved-Caspase3 in the combined treatment group (Figure 6E and 6F). In support of the mechanism identi ed in this study, the GSK3i group showed decreased BRCA1 protein level ( Figure  6E and 6F). All the tested compounds caused no obvious loss of weight of the nude mice ( Figure 6A and 6C) and were well tolerated during the drug administration.
To further validate the impact of GSK3β on in vivo sensitivity to PARPi, we used the HCT-15 GSK3β KO cells and parental cells to establish xenograft models in nude mice. As expected, administration of simmiparib signi cantly inhibited the growth of GSK3β KO tumor xenografts, but not the parental tumor xenografts. (Figure S5A and S5B). Consistently, there was a signi cant decrease in BRCA1 protein level and increase in γH2AX level in the GSK3β KO tumor xenografts treated with simmiparib ( Figure S5C). These data demonstrated that inhibition or depletion of GSK3β could enhance the in vivo sensitivity to simmiparib without toxicity.

Discussion
To identify effective drug combinations for BRCA-mutated cancer cells with de novo PARPi resistance, we tested the cellular effect of a panel of compounds either alone or in combination with PARPi in BRCA1mutated HCC1937 and BRCA2-mutated HCT-15 cells. Through this in vitro screen, we identi ed that a quarter of the oncological drugs and kinase inhibitors tested displayed synergy with PARPi in HCT-15 cells. These agents were included inhibitors of the DNA damage and cell cycle checkpoint (targeting ATR, CHK1 or CDK1), PI3K pathway (targeting PI3K, AKT or mTOR), and epigenetics regulators (targeting DNMT, HDAC and BET), and VEGFR. More importantly, the data suggested that GSK3 inhibition was most effective in enhancing the e cacy of PARPi. In conclusion, based on comprehensive and systematic screening of compounds, this study identi ed compounds that are capable of synergizing with PARPi.
Some of the synergistic interactions described in our screening were identi ed in previous studies [9, 10, 24-26, 28-30, 32]. For example, PARP inhibition was shown to synergize with: 1) PI3K pathway antagonism in BRCA-pro cient triple-negative breast cancer cells, 2) ATR-Chk1 inhibition in PARPiresistant BRCA-de cient cancer cells and high-grade serous ovarian cancer cells, 3) BET inhibition in multiple tumor lineages, 4) VEGFR antagonism in ovarian cancer cells. Our screens also revealed that the synergistic effect between PARPi and these compounds was far more prevalent in BRCA2-de cient HCT-15 cells (~ 25%) than in BRCA1-de cient HCC1937 cells (~ 4%), which implicated that populations with BRCA1 or BRCA2 mutations may bene t differently from PARPi-based combination therapies.
GSK3, a serine/threonine protein kinase with two functionally distinct isoforms, α and β, was discovered in the context of glycogen metabolism and has emerged as a ubiquitous regulator of multiple signaling pathways [34,35]. Historically, GSK3β has been thought of as a potential tumor suppressor due to its regulatory effect in the Wnt/β-catenin pathway [38]. However, large and ever increasing bodies of published data over the past decade have demonstrated that GSK3β is a positive regulator of cancer cell proliferation and survival in multiple tumor types [39]. CRC cells also displayed aberrant GSK3β expression and activity [40][41][42]. Direct pharmacologic inhibition of GSK3β signaling is, therefore, considered an attractive clinical strategy for these diseases [39]. A large number of GSK3i have entered clinical trials and several patent applications have been led and/or granted [43]. Unfortunately, GSK3i have shown limited bene ts, as monotherapy, in preclinical and clinical studies [44][45][46][47][48]. However, they appeared to be more effective when combined with other drugs [45,[49][50][51][52]. Here, our data showed strong synergistic effect between GSK3i and PARPi was observed in multiple CRC cell lines with diverse genetic backgrounds. Further in vivo studies showed that this new combination markedly suppressed tumor growth of HCT-15 and RKO tumor xenografts, without additional toxicity. Previous studies have demonstrated that olaparib combined with irinotecan displayed high toxicity concerns and no antitumor e cacy in CRC patients [53]. In this study, our results suggested that the combination of GSK3i and PARPi may produce encouraging responses with optimum tolerance in CRC patients.
In addition to regulating cellular processes including metabolism, growth, and survival, GSK3β also mediates the repair of DNA DSBs through phosphorylation of p53 binding protein 1 (53BP1) [54] and modulates the HRR pathway by phosphorylating the Fanconi anemia-associated protein (FAAP2), an important component of the Fanconi anemia complex involved in the repair of DNA interstrand crosslinks [55]. Furthermore, GSK3i altered the level of proteins involved in DNA repair, such as, ATR-interacting protein (ATRIP), topoisomerase IIβ binding protein (TopBP1) [51], tumor protein p53-induced nuclear protein 1 (TP53INP1) [52], and Tap63 [56]. In addition, GSK3β inhibition has been shown to enhance ionizing radiation-based sensitivity in vitro [57] and in xenograft models [54]. Our results advanced the current understanding of role of GSK3β by showing that GSK3β is essential for DSBs in HRR by affecting BRCA1 mRNA and protein expression. This mechanism was observed in all the cell lines with variable responses to the combination of GSK3 and PARP inhibition. However, the protein expression of BRCA1 was almost completely abrogated, while the mRNA level of BRCA1 only decreased to ~ 50% upon GSK3β inhibition and depletion, suggesting the involvement of other possible mechanisms. Therefore, the mechanism responsible for the suppression of BRCA1 expression by GSK3β remains to be further clari ed.

Conclusion
Collectively, our data provide a mechanistic understanding of combined PARP and GSK3 inhibition in CRC cells. Pharmacological and genetic studies suggested that loss of GSK3β activity impaired HRR e cacy, suppressed BRCA1 mRNA and protein levels and substantially sensitized cells to PARPi and Top I inhibitors in replication-dependent DSBs lesions. Our study implies that GSK3β is an important modulator of HRR. Notably, GSK3i may be combined with PARPi-based treatments in a wider population of CRC patients.

Declarations
This study was carried out in strict accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal studies were conducted in compliance with the Institutional Animal Care and Use Committee guidelines of the Shanghai Institute of Materia Medica (Shanghai, China).

Consent for publication
Not applicable and combination were de ned as IR1, IR2 and IR3, respectively. The color-coding denotes the level of IR (green [0% inhibition] to red [100% inhibition]) which was calculated as IR3-IR2-IR1. B and C, Effect of single agent and combination treatment on HCT-15 cell viability for combinations of PARP inhibitor, olaparib, plus CDK1 inhibitor RO-3306 (B) or ATR inhibitor VE-821 (C). OP, olaparib. Cell viability was measured by Sulforhodamine B assay (SRB). Combination index (CI) was calculated using CalcuSyn software with the Chou-Talalay equation, and average CI value are presented (CI < 1, synergism; CI = 1, additive effect; CI > 1 antagonism). Data are from three independent experiments and expressed as mean ± standard deviation (SD).  GSK3β depletion selectively sensitizes cancer cells to PARP and Top I inhibitors A and B, Levels of GSK3β and GSK3α protein in different GSK3β-/-or GSK3α-/-clonal variants (KO1 and KO2) of HCT-15 and RKO cells were detected using western blotting. C-E, Change in sensitivity to PARPi following GSK3β (C and D) or GSK3α depletion (E). Cells were treated with simmiparib (SP) and olaparib (OP) for 7 d then subjected