BKM120 Sensitizes Glioblastoma to the PARP Inhibitor Rucaparib By

Zhang et al. Cell Death and Disease (2021) 12:546 https://doi.org/10.1038/s41419-021-03805-6 Cell Death & Disease

ARTICLE Open Access BKM120 sensitizes glioblastoma to the PARP inhibitor rucaparib by suppressing homologous recombination repair Shaolu Zhang1,2,XinPeng1,XiaofeiLi1, Hongyan Liu2, Baoquan Zhao2, Moshe Elkabets 3, Yao Liu4,WeiWang4, Ran Wang1, Yuxu Zhong2 and Dexin Kong 1,5

Abstract PARP inhibitors have been approved for the therapy of cancers with homologous recombination (HR) deficiency based on the concept of “synthetic lethality”. However, glioblastoma (GBM) patients have gained little benefit from PARP inhibitors due to a lack of BRCA mutations. Herein, we demonstrated that concurrent treatment with the PARP inhibitor rucaparib and the PI3K inhibitor BKM120 showed synergetic anticancer effects on GBM U251 and U87MG cells. Mechanistically, BKM120 decreased expression of HR molecules, including RAD51 and BRCA1/2, and reduced HR repair efficiency in GBM cells, therefore increasing levels of apoptosis induced by rucaparib. Furthermore, we discovered that the two compounds complemented each other in DNA damage response and drug accumulation. Notably, in the zebrafish U87MG-RFP orthotopic xenograft model, nude mouse U87MG subcutaneous xenograft model and U87MG-Luc orthotopic xenograft model, combination showed obviously increased antitumor efficacy compared to each monotherapy. Immunohistochemical analysis of tumor tissues indicated that the combination obviously reduced expression of HR repair molecules and increased the DNA damage biomarker γ-H2AX, consistent fi

1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; with the in vitro results. Collectively, our ndings provide new insight into combined blockade of PI3K and PARP, which might represent a promising therapeutic approach for GBM.

Introduction 15 months2,3. Therefore, novel therapies for GBM are still Glioblastoma (GBM), the most malignant tumor of the urgently needed. adult central nervous system, represents up to 50% of all Poly (ADP-ribose) polymerase (PARP) is a nuclear primary brain gliomas1. Current GBM therapy, including protein known to function as a DNA damage sensor and aggressive surgical resection, high-dose external beam plays a role in DNA repair pathways via poly-ADP- radiation therapy (RT), and temozolomide (TMZ) che- ribosylating the automodification domain and enabling motherapy, is only associated with a median time to the recruitment of DNA repair proteins2,4. PARP inhibi- progression of 6 months and a median overall survival of tion ultimately causes double-strand break (DSB) accumulation during DNA replication and induces apoptosis, particularly in cells with homologous recombination (HR) Correspondence: Ran Wang ([email protected])or Yuxu Zhong ([email protected]) or Dexin Kong deficiency. This is the basis for the synthetic lethality of ([email protected]) PARP inhibitors (PARPis) in cancers with HR deficiency 1 Tianjin Key Laboratory on Technologies Enabling Development of Clinical due to mutations in BRCA1/2 or other HR genes5, Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin, China allowing these cancer cells to be selectively targeted while 2State Key Laboratory of Toxicology and Medical Countermeasures, Beijing sparing normal cells that have intact DNA repair sys- Institute of Pharmacology and Toxicology, Beijing, China tems6. Most recently, targeting autophagy was also Full list of author information is available at the end of the article These authors contributed equally: Shaolu Zhang, Xin Peng demonstrated to enhance the therapeutic efficacy of Edited by B. Joseph

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PARP inhibitor in HR proficient breast cancer cells7. Combination of rucaparib with BKM120 enhances Several PARPis have been approved for the therapy of apoptosis and DNA damage in GBM U251 and U87MG cells HR-deficient tumors8,9. However, since GBM is known to To determine the effect of the combination on apop- lack BRCA1/2 mutations and is, therefore, HR proficient, tosis in GBM cells, we next conducted Annexin V-PI PARPi alone might not exhibit optimal antitumor efficacy staining and flow cytometry analysis. Compared to each based on the synthetic lethality theory. The efficacy of drug alone, the combined treatment led to a significant PARPi was reported to be limited due to the super- increase in the late apoptotic population in U251 and activation of the PI3K-AKT signaling pathway10,11, and U87MG cells for 24 and 72 h (Fig. 2A, B and Suppl Fig. 2). loss of its counterpart PI3K-PTEN was found in Since DNA damage plays a key role in apoptosis, we next approximately 36% of GBMs11. Therefore, PI3K inhibitors determined whether the drug combination enhanced (PI3Kis) are expected to perform well in combination with DNA damage using an alkaline comet assay for detection PARPi to treat GBM. of both SSBs (single strand breaks) and DSBs in DNA. As BKM120 is a pan-PI3Ki in clinical trials for cancer ther- shown in Fig. 2C, D, compared to each single-agent apy12. Until now, pan-PI3Kis, including BKM120, have treatment, combination of the 2 drugs generated mark- shown modest benefits in the clinic because their doses for edly increased tail intensity in U251 and U87MG cells, therapy are limited due to toxicity13. Since drug combina- suggesting more severe DNA damage. Consistently, tions may enhance efficacy while minimizing toxicity, we combination treatment induced a dose-dependent recently examined the antitumor effect of the combination increase in the apoptosis marker cleaved PARP and the of PI3Ki BKM120 and the PARPi rucaparib on GBM DNA DSB marker γ-H2AX (Fig. 2E). in vitro and in vivo. We verified that BKM120 induced HR deficiency by downregulating the DNA repair process and Combination of rucaparib with BKM120 reduces HR repair sensitized BRCA-proficient tumor GBM to PARP inhibi- in GBM U251 and U87MG cells tion, providing novel insights into the treatment of GBM. Since HR plays a key role in the repair process of DNA DSB damage, we next investigated the effect of the Results combination on HR repair. First, an HR reporter assay Combination of BKM120 and rucaparib exhibits a showed that HR repair efficiency was reduced after synergetic antiproliferative effect on GBM U251 and cotreatment of rucaparib with BKM120 (Fig. 3A, B). U87MG cells Compared to rucaparib alone, cotreatment with BKM120 We first examined the antiproliferative effect of reduced expression of the HR repair molecules BRCA1, BKM120 and rucaparib using an MTT assay. As shown in BRCA2, and RAD51 at both the protein and mRNA levels Fig. 1A, either of the two compounds alone inhibited the (Fig. 3C, D). Consistently, immunofluorescence analysis proliferation of GBM U251 and U87MG cancer cells in a indicated that the number of RAD51 foci was significantly dose-dependent manner, with IC50 values of 0.90 and reduced while the number of γ-H2AX foci was increased 1.38 μM (BKM120) and 14.36 and 15.00 μM (rucaparib), in U251 and U87MG cells in the combination of the 2 respectively. Next, we determined the antiproliferative drugs compared to each drug alone (Fig. 3E, F). In addi- effect of the combination of BKM120 and rucaparib (with tion, rucaparib increased expression of RRM2 (Ribonu- ratios of 0.5 vs 10 μM for U251 cells and 1 vs 10 μM for cleotide Reductase small subunit M2) and U87MG cells, the same as the following in vitro experi- phosphorylation of ATR and CHK1 (checkpoint kinase 1), ments) and analyzed their combinational effect using and these phenomena were reversed by cotreatment with Chou-Talalay’s method14. As indicated in Fig. 1B and BKM120. On the other hand, BKM120 alone induced Tables 1 and 2, all the combination index (CI) values were increased poly-ADP-ribosylation (PAR), which was less than 1, suggesting that the combinations were reversed by cotreatment with the PARPi rucaparib synergetic. Next, we performed colony formation assays (Fig. 3C). to determine the effect of the combinations on the tumorigenic ability of GBM cells. The colony formation of BKM120 enhances the antitumor efficacy of rucaparib in a U251 and U87MG cells with a higher potency than either zebrafish U87MG orthotopic xenograft model drug alone (Fig. 1C, D). As cell cycle progression is To evaluate the in vivo anti-GBM efficacy of the com- essential for cell proliferation, we investigated the effect of bination, we first determined the effect on a zebrafish this treatment combination on the cell cycle by flow U87MG orthotopic xenograft model. Tumor burden was cytometry analysis. Figure 1E, F and Fig. S1 indicated that evaluated by measuring the integrated RFP fluorescence treatment with rucaparib for 24/48/72 h led to G2/M cell intensity. As shown in Fig. 4A, B, BKM120 alone led to an cycle arrest, but the effect was attenuated when treated in ~50% reduction compared to the vehicle group. When the presence of BKM120. combined with rucaparib, tumor growth was further

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Fig. 1 The in vitro antiproliferative activities of BKM120 and/or rucaparib in human GBM cells. A U251 and U87MG cells were treated with various concentrations of BKM120 and rucaparib (BKM-120 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 25 μM; rucaparib-0.2, 0.5, 1, 2, 5, 10, 25, 50, 100 μM), and antiproliferative effects were determined by MTT assay following 96 h incubation. B Cells were treated with BKM120 and rucaparib as single agents or in combination for 96 h and then subjected to MTT assay. The drug combination effects were analyzed using CalcuSyn software, and the resulting CI-Fa plots are shown. C U251 cells were treated with 0.5 μM BKM120 or/and 10 μM rucaparib, and U87 cells were treated with 1 μM BKM120 or/and 10 μMrucaparib (the same concentrations were used in subsequent experiments unless indicated) for 10 days and then stained with crystal violet. D Colonies with 50 or more cells were quantiﬁed using ImageJ. E Cells were treated with BKM120 and/or rucaparib for 24/48 h. After PI staining, ﬂow cytometry analysis was performed to determine the cell cycle distribution. F Cell cycle distribution of the cells exposed to the single drugs or combination are represented as stacked columns. Statistical analysis of G2/M phase in each group. All data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

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Table 1 Combination indexes (CI) of BKM120 and after administration of the combination, consistent with Rucaparib in U251 cells. the in vitro results (Fig. 5H).

Drug or drug combination r CI values BKM120 enhances the antitumor efficacy of rucaparib in a nude mouse U87MG orthotopic xenograft model ED50 ED75 ED90 Finally, we used an U87MG orthotopic nude mouse BKM120 (0.5 μM) 0.96990 —— xenograft model to examine whether combination of the two drugs could exhibit anti-GBM efficacy after passing Rucaparib (10 μM) 0.97739 ——— the blood brain barrier (BBB), which is known as a barrier Combo 0.99574 0.45711 0.50727 0.67014 for water-soluble drugs. U87MG cells stably expressing luciferase (U87MG-Luc) were used to facilitate live imaging of tumor growth. Ten days postinjection of Table 2 Combination indexes (CI) of BKM120 and U87MG-Luc cells in the corpus striatum of mice, mice Rucaparib in U87MG cells. were subjected to drug treatment. After 14 days of treatment, either BKM120 or rucaparib exhibited sig- Drug or drug combination r CI values nificant antitumor effects as a single agent. When combined together, tumor burden was further significantly ED50 ED75 ED90 reduced compared to each drug alone (Fig. 6A, B). BKM120 (1 μM) 0.95250 —— However, both BKM120 and the combination treatment caused a reduction in body weight, suggesting potential Rucaparib (10 μM) 0.93404 ——— toxicity (Fig. 6C). The brain tissue stained with hema- Combo 0.99831 0.66068 0.53476 0.46661 toxylin and eosin showed that U87MG orthotopic xenografts grew in a noninvasive way, and tumors from mice administered combination treatment were smaller inhibited, with an ~80% reduction compared to the than tumors from the other groups (Fig. 6D). Immuno- vehicle group. histochemical analysis indicated downregulation of BRCA1/2 and Rad51 and upregulation of γ-H2AX in BKM120 enhances the antitumor efficacy of rucaparib in a response to the combination of rucaparib and BKM120 nude mouse U87MG subcutaneous xenograft model compared to rucaparib alone, consistent with the in vitro We subsequently investigated the antitumor efficacy of results (Fig. 6E, F). the drug combination using a nude mouse U87MG subcutaneous xenograft model. BKM120, rucaparib, the Discussion combination, and the vehicle were administered to the PARPis have exhibited convincing antitumor efficacy on animal models. Tumor volume and body weight were BRCA-deficient ovarian and breast tumors13. In GBM, measured every 3 days. Sixteen days later, mice were where BRCA1/2 functional mutations/deletions are rare sacrificed, and tumors were excised for tumor weight (≤1%)15, PARPi merely exerts strong anti-GBM effects in measurement. As indicated in Fig. 5A–D, treatment with a few cases, such as those with IDH1/2 mutations16. BKM120 or rucaparib as single agents led to robust However, in most cases, BRCA1/2 can be recruited to inhibition of tumor growth both in volume and in weight. damaged DNA for the repair of DSBs, reducing the When administered in combination, tumor growth was antitumor efficiency of PARPis17. On the other hand, further inhibited compared to each drug alone. By the end PI3K was reported to stabilize and preserve DSB repair by of the 16-day treatment period, the combination led to an interacting with the HR complex18. Combination thera- over 90% reduction in both tumor volume and tumor pies with PARPi and PI3Ki have shown considerable weight. However, the combination also caused a slight promise for the treatment of ovarian cancer19,20, triple- decrease in body weight compared to each drug alone and negative breast cancer21,22 and prostate cancer23. In this to the vehicle group (Fig. 5E). Tumor tissues of each study, we found that PI3Ki BKM120 induced HR defi- group were used for H&E, immunostaining and western ciency by downregulating the DNA repair process, sen- blotting. As shown in Fig. 5F, G, the number of cells sitizing BRCA-proficient GBM to PARPi. stained with Ki67, a biomarker of cell proliferation, was We demonstrated that combination of the PARPi reduced after treatment with each drug alone or in rucaparib with the PI3Ki BKM120 resulted in synergetic combination, and the combination further inhibited inhibition of GBM cell proliferation. Cell cycle analysis U87MG proliferation compared to each drug alone. indicated that rucaparib alone arrested the cell cycle in Western blot analysis showed that expression of γ-H2AX G2/M phase, but when combined with BKM120, the was increased, while BRCA1/2 and RAD51 was decreased effect was weakened. Meanwhile, apoptosis and DNA

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Fig. 2 BKM120 and rucaparib cooperate to induce apoptosis and DNA damage in GBM cells. U251 and U87MG cells were treated with BKM120 and rucaparib as single agents or in combination. A The percentage of apoptotic cells was determined by Annexin V and PI staining at 72 h. B FACS quantification of the respective cell population including Annexin V + /propidium iodide− early apoptotic cells, and Annexin V + /propidium iodide+ late apoptotic cells. C DNA damage in GBM cells was determined by alkaline comet assay to detect both SSBs and DSBs for 12 and 24 h. Comet images × 200 acquired by fluorescence microscopy are shown. Scale bar: 100 μm. D The tail moment was used to quantify DNA damage with CASP software. E The expression levels of the apoptosis marker PARP (cleaved and full length) and the DNA DSB marker γ-H2AX were determined by western blotting at 48 h. All data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. damage were most serious in cells treated with the com- attenuation of G2/M arrest may also be regarded as bination. It was reported that PARPi activates the G2/M enhancement of G1 arrest. checkpoint and causes cell cycle arrest in G2/M until We also found that BKM120 combined with rucaparib DNA damage was repaired24. Therefore, BKM120 inhib- induced a marked increase in DSBs, one of the most ited DNA damage repair and eventually led to apoptosis, harmful lesions to cells15,25. It is well known that the HR which might be related to the attenuation of the G2/M pathway plays a major role in the late S and G2 phases to checkpoint arrest caused by rucaparib. However, this repair DSBs, so we examined whether BKM120 enhanced postulation remains to be verified since the above the effect of rucaparib by regulating HR. The HR reporter

Official journal of the Cell Death Differentiation Association , C Cells F , E ciency. fi Page 6 of 14 -H2AX in GBM cells γ * * ** -+ * -+ -+ -+ ** Rucaparib Rucaparib Rucaparib Rucaparib *** *** ). All data are presented as Ctrl Ctrl Ctrl Ctrl U87MG-12 h Quantitative reverse U87MG-24h U87MG-24 h F -+ -+ U87MG-12 h -+ -+ D * BKM120 BKM120 BKM120 BKM120 γ-H2AX RAD51 45 kDa 60 kDa >200 kDa >200 56 kDa 60 kDa 37 kDa 300 kDa 45 kDa 56 kDa 300 kDa 380 kDa 220 kDa * * ** ** -+ * -+ * -+ -+ Rucaparib Rucaparib Rucaparib Rucaparib + + *** *** U251-24 h U251-24h - + Ctrl U251-12 h Ctrl U251-12 h Ctrl Ctrl -+ -+ -+ -+ * - + U87MG U251 and U87MG cells were transfected with DR- F BKM120 BKM120 BKM120 BKM120 - - 1.00 0.36 1.04 0.10 uorescence staining of RAD51 and -H2AX foci was shown in ( γ fl Combo + + Combo - + ** U251 - + Cells were collected for western blotting detection of DNA damage- - - Rucaparib 1.00 1.00 0.98 1.40 0.85 1.00 0.63 1.14 0.39 Rucaparib 1.00 1.00 0.70 1.32 0.79 1.00 0.38 1.36 0.41 1.00 0.20 0.76 0.45 1.00 0.31 0.90 0.55 C 1.00 1.62 0.29 0.36 0.29 1.62 1.00 1.00 4.00 0.94 0.41 The percentage of GFP-positive cells represents HR repair ef 1.00 1.30 2.00 1.51 2.00 1.30 1.00 1.00 0.56 0.76 0.63 1.00 1.03 1.03 1.09 1.00 1.08 1.02 1.10 1.00 1.17 1.01 1.01 1.17 1.00 1.10 0.98 1.00 0.99 0.99 1.00 0.83 1.17 0.96 1.17 0.83 1.00 0.76 1.00 1.45 1.80 1.00 0.32 0.96 0.12 1.00 1.041.00 0.95 0.99 1.00 1.03 1.00 1.10 1.00 1.07 0.29 0.90 0.29 1.00 1.00 0.62 B

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5 4 3 2 1 0 12h 105 104 103 102 101 100 10 10 10 10 10 10 105 104 103 102 101 100 105 104 103 102 101 100 24 h 5 5 5 5 10 10 10 10 4 + 4 4 4 U87MG 10 10 10 10 7.6% 0.3% 3 7.0% 3 3 3 <0.1% 10 10 Ctrl 10 10 2 2 - + 2 2 - 10 FITC-A 10 m. The percentage of cells with RAD51/ ** FITC-A FITC-A 10 10 1 1 1 Combo 1 Combo 10 10 μ 10 10 0 0 0 0 10 10 10 10 105 104 103 102 101 100 105 104 103 102 101 100 105 104 103 102 101 100 105 104 103 102 101 100 5 5 5 5 10 10 10 10 4 4 4 < 0.001. 4 BKM120 10 10 10 10 3 3.5% 3 3 3 0.1% 2.9% 0.3% P 10 10 10 10 2 2 2 2 - + + 10 10 10 10 FITC-A FITC-A 1 1 1 1 Rucaparib 10 10 10 10 (2021) 12:546 (2021) 12:546 Rucaparib 0 0 0 0 *** 10 10 10 10 - 105 104 103 102 101 100 105 104 103 102 101 100 105 104 103 102 101 100 105 104 103 102 101 100 Rucaparib 5 5 5 5 U251 10 10 10 10 U251 * 4 4 4 4 U251 10 10 10 10 + 3 5.8% 3 3 0.3% 3 5.7% <0.1% < 0.01; *** 10 10 10 10 2 2 2 2 - - Ctrl 10 10 10 10 FITC-A FITC-A P 1 1 1 1 - BKM120 BKM120 10 10 10 10 0 0 0 0 10 10 10 10

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DAPI DAPI γ-H2AX RAD51 Merged γ-H2AX RAD51 Merged A B E 3). * D = n Cell Death and Disease U251 and U87MG cells were treated with BKM120 and/or rucaparib for 48 h. cial journal of the Cell Death Differentiation Association ﬁ mean ± SD ( Cell nuclei were stained with DAPI. Scale bar: 20 transcription PCR analysis of BRCA1, BRCA2, and RAD51 expression in two cancer cell lines. Gene expression was normalized to 18 s rRNA. were treated with BKM120 or/and Rucaparib for 12/24 h. Representative images of immuno D and HR repair-related proteins, including BRCA1, BRCA2, RAD51, RRM2, p-ATR, p-CHK1, p-AKT, ATR, CHK1, and PAR. GFP plasmid and pCBASceIresuspended plasmid in using ice-cold Lipofectamine PBS. GFP 3000. intensity Cells was were analyzed by treated with BKM120 or/and rucaparib for 72 h and then collected and Fig. 3 Effects of BKM120 and rucaparib as single agents or in combination on HR repair. A Of Zhang et al. Zhang et al. Cell Death and Disease (2021) 12:546 Page 7 of 14

Fig. 4 BKM120/rucaparib combination treatment inhibits tumor growth in the U87MG zebrafish xenograft model. A After transplantation with 50–100 U87MG-RFP cells (red fluorescence), the injected embryos were transferred to a 96-well plate containing drug and incubated for 96 h. The embryos were imaged with a fluorescence microscope to detect tumor growth. Scale bar: 100 μm. B The relative increase in fluorescence intensity was determined by dividing the measured values by the fluorescence intensity of the tumor in the same embryo at 0 days after implantation. Student’s t-test was used to compare the effect of each treatment with vehicle or treated animals. *P < 0.05; **P < 0.01; ***P < 0.001. assay revealed that HR repair efficiency was reduced after PI3K may be a plausible strategy for the treatment of cotreatment of rucaparib and BKM120 compared to GBM18. BKM120 induced an increase in PAR, which was rucaparib alone. BKM120 also inhibited the transcription reversed by cotreatment with rucaparib, as shown in both and translation of the HR molecules BRCA1 and BRCA2. in vitro and in vivo experiments (Fig. 3B and Fig S3). Ibrahim et al reported that PI3K inhibition resulted in the Consistent with our result, BKM120 was reported to compensatory activation of MAPK/ERK pathway, thus upregulate PAR as well as H2AX in BRCA1-related breast phosphorylated and activated ETS (E26 Transformation- cancer21. Suppression of the PI3K pathway was accom- specific Sequence) transcription factors to reduce BRCA1/ panied by cleaved PARP upregulation and an increase in 2 transcription through repressing the BRCA1/2 pro- PARP activity (increased PAR levels), suggesting that cells moter, eventually induced HR repair deficiency in triple- undergoing PI3K suppression may be recovered by PARP- negative breast cancer cells21. In addition, RRM2, a dependent DNA repair. GBM cells widely express PARP- downstream effector of BRCA1, was also reduced after 1; therefore, targeting PARP-1 might represent an rucaparib/BKM120 treatment. Rismussen et al. reported appropriate strategy for overcoming apoptotic that BRCA1 loss impedes replication fork progression by resistance30,31. downregulating RRM2 in GBM cells17. Furthermore, We used three types of subcutaneous and orthotopic BKM120 treatment decreased Rad51 expression and foci xenograft models32,33 to evaluate the in vivo anti-GBM formation, which might be attributed to the inactivation effect of the BKM120/rucaparib combination. Consistent of AKT, as AKT inactivation was reported to inhibit with the in vitro studies, the combination obviously Rad51 expression26,27. Besides, the ATR-CHK1 pathway is enhanced the antitumor efficacy in all U87MG xenograft the principal effector of DNA damage and is essential for models compared to each drug alone. Western blotting HR. ATR signaling was also demonstrated to be involved and IHC analysis of tumor tissues indicated that rucaparib in PARPi resistance28. Kumar et al found that PI3K-p110β increased expression of the HR repair effectors BRCA1/2 played a key role in DSB regulation. Knockdown of and Rad51, while these molecules were reduced by the PIK3CB gene inhibited the recruitment and phosphor- combination, suggesting that BKM120 sensitizes tumor ylation of ATR, and subsequently the phosphorylation of tissues to the effect of rucaparib by suppressing HR repair CHK1, thus increased genomic instability29. Therefore, we in vivo. Failure to cross the blood–brain barrier (BBB) is investigated the effect of rucaparib/BKM120 on ATR and the bane of drug development for cerebral disease. Similar CHK1 and found that BKM120 attenuated phosphoryla- to many other drugs, rucaparib has been proven to be a tion of ATR and CHK1, inhibiting HR and sensitizing substrate of efflux transporters in brain endothelial cells; cells to rucaparib. thus, the ability of rucaparib itself to pass through the BBB Notably, we discovered that BKM120 and rucaparib is limited34,35. Recent studies have shown that PI3Ki can complemented each other during the DNA damage increase drug accumulation in cells by inhibiting the response. Since tumor resistance is frequently reported to expression of efflux transporter protein P-gp and/or be mediated by activation of the PI3K pathway, targeting BCRP36,37. We found that rucaparib increased the

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Fig. 5 BKM120/rucaparib combination treatment markedly reduced tumor growth in U87MG nude mouse heterotopic xenograft models. A–H Nude mice with subcutaneous U87MG tumors were treated with vehicle, BKM120 (15 mg/kg), rucaparib (4 mg/kg), or the BKM120/rucaparib combination for 16 days (n = 7). After sacriﬁcing mice, images were taken as shown in (A). Then, tumors were taken from mice with the pictures indicated in (B). C Measurement of tumor weight. Time-course measurements of tumor volumes (D) and body weights (E) every 3 days. F H&E and Ki67 immunohistochemistry staining of tumor tissues from U87MG xenograft mice treated with BKM120 and/or rucaparib. Scale bar: 50 μm. G Statistical quantiﬁcation of (F) shows U87MG cell proliferation in xenografts with the indicated treatments. H western blotting analysis of BRCA1 and γ-H2AX in harvested tumors. Data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

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Fig. 6 BKM120/rucaparib combination treatment inhibits tumor growth in U87MG nude mouse orthotopic xenograft models. A–F Mice were inoculated with U87MG-Luc cells. The localization and intensity of luciferase expression were monitored by in vivo bioluminescence imaging (dpi, days post cell injection). Following a single dose of BKM120 (30 mg/kg) or rucaparib (10 mg/kg) or combination treatment of BKM120 and rucaparib for 14 days (n = 5), animals were measured by a bioluminescence imaging system. A Representative images of mice taken by the bioluminescence imaging system. B The relative radiance, which represents tumor size, was determined by the imaging system. C Body weights were monitored during the 14 days of treatment. D Hematoxylin and eosin-stained section (original magnification ×20) of intracranial U87MG mouse xenografts following BKM120 and/or rucaparib treatments. Scale bar: 1 mm. E Representative immunohistochemistry staining for BRCA1/2, γ-H2AX, and RAD51 in tumor tissues of U87MG xenografts. Scale bar: 50 μm. F Statistical quantification of (E) shows HR repair efficiency and DNA DSB levels in U87MG xenografts with the indicated treatments. Data are presented as the mean ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

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expression of BCRP and P-gp in brain tissues, which could were purchased from Abcam. pDR-GFP, pCBASce-I be suppressed by cotreatment with BKM120. And plasmids, and pEGFP-C1 were kindly provided by Dr. BKM120 alone could downregulate the two transporters François X. Claret (MD Anderson Cancer Center, as well, especially BCRP (Fig. S3). Therefore, in addition Houston, TX). to inhibiting HR, we speculated that BKM120 might sensitize the in vivo antitumor effect of rucaparib by Cell proliferation assay increasing its effective concentration in the tumor area. Cell proliferation was measured by MTT assay as we However, it should be noted that the body weight of nude previously described38. The cells were seeded at a density mice treated with the combination was slightly lower than of 5 × 103 cells/ml in a volume of 200 μl/well in 96-well those treated with BKM120 or rucaparib alone, which plates. The next day, cells were treated with DMSO or a might be due to combined cytotoxicity. Therefore, series of concentrations of BKM120 or rucaparib. Four appropriate doses of the two compounds should be days later, 20 μl of MTT (5 mg/ml) was added to each characterized in early clinical trials, and sequential ther- well. After further incubation for 4 h at 37 °C, the absor- apy might be considered to reduce toxicity. bance at 490 nm was measured using an iMark microplate Taken together, we demonstrated that combined reader (BIO-RAD, Hercules, CA, USA). administration of the PI3Ki BKM120 and the PARPi rucaparib is effective at inhibiting GBM tumor growth Drug combination analysis both in vitro and in vivo. Cotreatment with BKM120 and First, the MTT assay was performed as described in 2.2. rucaparib synergistically induces apoptosis and DNA The half-maximal inhibitory concentration (IC50) values damage in GBM cells. PI3K inhibition by were acquired from dose-response curves utilizing BKM120 suppresses HR, conferring sensitivity to ruca- GraphPad Prism 7 software. Then, drug synergy was parib. Our study suggests that combined blockade of PI3K analyzed based on Chou and Talalay’s equation14. Briefly, and PARP might be a promising therapeutic approach for three cell lines were treated for 4 days with BKM120 or/ GBM, but further investigations are needed to confirm and rucaparib at a constant concentration. Data obtained these findings. from the growth inhibitory experiments were analyzed by CalcuSyn software to determine the drug combination Materials and methods effect. Combination indexes (CI) were then calculated, Cell culture and reagents with CI < 1, CI = 1 and CI > 1 indicating synergism, U251 and U87MG cell lines were purchased from Cell additivity, and antagonism, respectively. Resource Center, Peking Union Medical College (Beijing, China). Cells were cultured in RPMI 1640 (for U251) or Colony formation assay DMEM (for U87MG) supplemented with 10% FBS and The colony formation assay was performed as pre- 1:100 penicillin/streptomycin (Invitrogen, Carlsbad, CA, viously described39. U251 and U87MG cells were seeded USA) at 37 °C in 5% CO2. Cell lines were authenticated by into 24-well plates at 500 cells/well and then treated with STR profiling and confirmed to be without mycoplasma BKM120 and/or rucaparib or vehicle control (DMSO) the contamination. next day. Cell culture medium containing drug or vehicle BKM120 and rucaparib were purchased from Selleck was changed every three days. After incubation for (Danvers, MA, USA). MTT reagent was purchased from 10 days, colonies were fixed and stained with 0.25% crystal Amresco (Solon, OH, USA). The Annexin V-FITC/PI violet (Sigma-Aldrich, St. Louis, MO, USA) for visuali- Apoptosis Detection Kit was purchased from BD Bios- zation. Colonies with 50 or more cells were counted using ciences (San Jose, CA, USA). TRIzol reagent was pur- ImageJ software. chased from Life Technologies (Carlsbad, CA, USA). Star Script II First-strand cDNA Synthesis Mix and 2× Real- Flow cytometry Star Green Fast Mixture were purchased from GenStar Detection of apoptosis was performed using an Annexin (Beijing, China). Antibodies against RAD51 (#385533), V-FITC/PI Apoptosis Detection Kit (BD Biosciences) as BRCA-1 (#342823), BRCA-2 (#160133), ATR (#161471), previously described40. Briefly, U251 and U87MG cells CHK1 (#380200) were purchased from zen-bio (Chengdu, were seeded into 6-well plates at a density of 40% con- China), caspase-3 (#9662), BRCA-1 (#9010), BRCA-2 fluence. The next day, cells were exposed to BKM120 (#10741), PARP (#9532), p-ATR (#2853), p-CHK1 and/or rucaparib. After 24/72 h of treatment, cells were (#2347), RRM2 (#65939), PAR (#83732) ABCB1 collected and suspended in binding buffer containing (#13978), ABCG2 (#42078), γ-H2AX (#80312), and Ki67 Annexin V-FITC. The cell suspension was further incu- (#9449) antibodies were purchased from Cell Signaling bated with propidium iodide (PI) for 15 min. Apoptosis Technology. Alexa Fluor 488 (#ab150113)- and Alexa was detected by FACS Verse Flow Cytometry (BD Bios- Fluor 594 (#ab150080)-conjugated secondary antibodies ciences) and quantified using Flow Jo Software (Tristar,

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Ashland, OR, USA). The percentage of cells in the upper 20 min. After mounting, coverslips were immunostained right (representing late apoptotic cells) and lower right with mouse monoclonal anti-γ-H2AX or rabbit polyclonal quadrants (representing early apoptotic cells) were sum- anti-RAD51 overnight at 4 °C. The next day, coverslips med to obtain the percentage of apoptotic cells. were washed and incubated using the respective second- For analysis of cell cycle distribution, cells were seeded ary antibody (1:50) at room temperature for 1 h. Nuclei into 6-well plates and treated with the indicated drugs on were counterstained using DAPI (Solabio, Beijing, China). the following day as reported in our previous study41. An Olympus FV1000 scanning confocal microscope After 24/48/72 h of incubation, cells were collected, sus- (Toyko, Japan) was used to capture images. The γ-H2AX pended in PBS, and fixed in 75% ethanol at 4 °C overnight. and RAD51 foci were counted from at least 50 cells per Next, cells were washed and resuspended in PBS con- sample. taining 50 μg/ml PI and 100 μg/ml RNase (Solabio, Beij- ing, China). All samples were analyzed on a FACS Verse Homologous recombination assay Flow Cytometer (BD Biosciences). HR efficiency was evaluated as reported in a previous study44. U251 and U87MG cells were seeded into 6-well Alkaline comet assay plates and cultured overnight. The HR repair reporter After treatment with BKM120 or/and rucaparib for 12/ substrate pDR-GFP plasmid and the pCBASceI plasmid 24 h, U251 and U87MG cells were collected and subjected were transfected into cells using Lipofectamine 3000 to alkaline comet assay for detection of both SSBs and (Thermo Fisher, Illkirch, France). GFP-expressing plasmid DSBs as we described previously42. The cells (1 × 105/ml) (pEGFP-C1) was used as a transfection efficiency control. were combined with molten LMAgarose at a ratio of 1:10 Cells were treated with BKM120 and/or rucaparib for (v/v), and 50 µl of cell suspension was immediately added 48 h. Then, cells were harvested and resuspended in ice- onto comet slides. The slides were then incubated at 4 °C cold PBS, and GFP-positive cells were detected using a for 10 min, immersed in lysis solution for 30 min, and in FACS Verse flow cytometer (BD Biosciences). alkaline unwinding solution for 20 min in the dark. Fol- lowing electrophoresis, the cells were stained with ethi- Quantitative real-time PCR dium bromide (EB). Approximately 200 individual cell qRT-PCR analysis was performed as previously repor- images of each group were analyzed using CASP software ted45. U251 and U87MG cells were treated with BKM120 (CaspLab, Wroclaw, Poland). Tail DNA (percentage of and/or rucaparib for 48 h. Total RNA was isolated from DNA in the tail) was calculated from at least 50 cells of cells using TRIzol according to the manufacturer’spro- each group as an indicator of DNA damage. tocol. cDNA was synthesized from 1 μg of total RNA using StarScript II First-strand cDNA Synthesis Mix (Genstar), Western blotting and RT-PCR was performed with aliquots of cDNA sam- Western blot analysis was performed as described in a ples mixed with 2× RealStar Green Fast Mixture. Results 43 previous study . U251 and U87MG cells were treated are expressed as the fold change calculated by the ΔΔCt with BKM120 and/or rucaparib for 48 h. Then, cells were method relative to the control sample. The ribosomal harvested and lysed in RIPA buffer supplemented with 1% subunit 18 S was used as an internal normalization control. phosphorylation inhibitors and 1% protease. Total protein Sequences of the PCR primers were as follows: BRCA1 Fw (50 µg) for each sample was loaded onto an SDS-PAGE 5′-GAACGGGCTTGGAAGAAAAT-3′ and Rv 5′-GTTT gel. After electrophoresis, proteins were transferred onto CACTCTCACACCCAGA-3′. BRCA2 Fw 5’-CAGGTAG polyvinylidene fluoride membranes (BIO-RAD). After ACAGCAGCAAGCA-3’;Rv5’-AAGCCCCTAAACCCCA blocking in 5% nonfat dried milk, membranes were CTTC-3’. RAD51 Fw 5’-CAGATGCAGCTTGAAGCAA incubated with primary antibodies overnight at 4 °C and A-3’;Rv5′-TTCTTCACATCGTTGGCATT-3′.18S then washed and incubated with HRP-conjugated sec- rRNA Fw 5′- CAGCCACCCGAGATTGAGCA-3′;Rv5′-T ondary antibodies for 1 h. Expression of target protein was AGTAGCGACGGGCGGTGTG-3′. visualized using an ECL detection kit (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). U87MG orthotopic zebrafish model Xenotransplantation of human glioblastoma U87MG Immunofluorescence staining cells and proliferation assessment were performed as Immunofluorescence staining was performed as described in a previous study33. Wild type AB zebrafish described in our previous report42. U251 and U87MG (Daniorerio) were maintained and reared on a 14 h light- cells were cultured on coverslips in 24-well plates in the 10 h dark cycle at 28 °C in a controlled multitank recir- respective media containing inhibitors for 12/24 h. Then, culating system. Embryos were collected and incubated in cells were fixed with 4% paraformaldehyde, diluted with reconstituted water (60 µg/ml sea salt in RO water with 1 PBS, and permeabilized with 0.5% Triton X-100 buffer for ppm methylene blue). At 48 h postfertilization, embryos

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were anesthetized using 1.2 mM tricaine and moved onto mice were sacrificed by using excessive pentobarbital a modified agarose gel mold for tumor cell microinjection. sodium. Data were analyzed using in vivo imaging A total of 50–100 U87MG-RFP cells suspended in 5 nl of software. serum-free culture medium were injected into the brains of zebrafish larvae via a pneumatic pico-pump injector. Examination of the compounds in brain tissues of non- After cell implantation, injected embryos were screened tumor mouse and separately transferred to a 48-well plate containing We used non-tumor mouse to evaluate the effect of drugs in 2 ml of E3 media and incubated at 32 °C for rucaparib and BKM120 alone or in combination on 4 days. Xenografts were observed with an Olympus (IX71) PARylation and efflux transporters in the brain tissue, to inverted microscope every 2 days. The fluorescence verify that the 2 compounds passed through BBB. Four- intensity of xenografts was quantified using ImageJ five weeks BALB/C nude mice were treated as follows: software. vehicle, BKM120 (30 mg/kg), rucaparib (10 mg/kg), or BKM120/rucaparib combination (with the same doses as U87MG heterotopic nude mouse model each single agent) for 3 days. Then, the mice were killed All animal experiments were performed in accordance by using excessive pentobarbital sodium and the brain with the National Institutes of Health Guide for the Care tissue was removed. Half of the tissue was formalin-fixed and Use of Laboratory Animals (Ethical approval No.: and paraffin-embedded for histological analysis, and the IACUC-DWZX-2020-753. Date: 25 August 2020 to 25 other half was snap-frozen in liquid nitrogen for western October 2020). To generate a murine subcutaneous tumor blotting. model, U87MG cells (1 × 107 cells per mouse) were injected subcutaneously into the right lateral flank of 4- to 5-week- H&E and immunohistochemical (IHC) staining oldmaleBALB/Cnudemice(VitalRiverLaboratoryAnimal H&E and IHC staining were performed as previously Technology Company, Beijing, China). When tumors reported46 with a small modification. H&E staining was reached a volume of 800–1000 mm3, tumor masses were used to detect pathological changes. For histological divided into 2 × 2 × 2 mm3 masses and implanted into the analysis, formalin-fixed, paraffin-embedded tumors were right flankof28BALB/cnudemice.Tumorswereallowed sectioned, and slides were deparaffinized using xylenes to grow to a volume of 100 mm3, and then the animals were (Thermo Fisher Scientific Inc.). Endogenous peroxidases randomly divided into four groups. Each group of 7 mice were quenched with 3% hydrogen peroxide in methanol. was treated with vehicle, BKM120 (15 mg/kg, po.), rucaparib Staining was performed using antibodies against BRCA1 (4 mg/kg, ip.), or BKM120 and rucaparib (with the same (1: 100), BRCA2 (1: 100), RAD51 (1: 50), γ-H2AX (1: 50), doses as a single agent). Tumor size was measured every P-gp (1: 200), BCRP (1: 200), PAR (1: 250), or Ki67 (1: three days until the endpoint, and the volume was calculated 500). Counterstaining was performed using Mayer’s using the formula (length × width2)/2. At the end of the hematoxylin (Dako, Glostrup, Denmark). Images 16 days, the mice were sacrificed by using excessive pento- were observed under an Olympus Cx21 microscope, barbital sodium, and then the tumors were removed. Half of scanned with a high-resolution digital slide scanner the tumor tissues were formalin-fixed and paraffin- (Pannoramic 250, 3DHistech), and quantified using embedded for histological analysis, and the other half of ImageJ software. tumors were snap-frozen in liquid nitrogen for western blotting. Statistical analysis All results were repeated in triplicate unless otherwise U87MG orthotopic mouse model stated, and quantitative data are expressed as the mean ± To generate a murine intracranial tumor model, standard deviation (SD). Statistically significant differ- U87MG-Luc cells (2.0 × 105 cells per mouse) were injec- ences between pairs of mean values were determined by ted into the right striatum (1 mm lateral to bregma, Student’s t-test. Statistical significance was defined as a 0.8 mm anterior to bregma, and 3 mm deep from the P-values < 0.05. dura) of 4- to 5-week-old male BALB/C nude mice. Nine days later (Day 10), animals were measured using an IVIS Acknowledgements This work was supported by grants from the National Natural Science luminescent imaging system (IVIS Spectrum, Perki- Foundation of China (81673464, 82073890, and 8201101056) and a grant for nElmer) and randomly divided into four groups. The the Major Project of Tianjin for New Drug Development (17ZXXYSY00050). respective groups of mice were treated as follows: vehicle, BKM-120 (30 mg/kg), rucaparib (10 mg/kg), BKM-120, Author details 1 and rucaparib (with the same doses as each single agent). Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, After treatment for 7 and 14 days, animals were examined Tianjin, China. 2State Key Laboratory of Toxicology and Medical by luminescence imaging. At the end of the 14 days, the Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing,

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China. 3The Shraga Segal Department of Microbiology, Immunology and 13. Konstantinopoulos, P. A. et al. Olaparib and α-specific PI3K inhibitor alpelisib Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer- for patients with epithelial ovarian cancer: a dose-escalation and dose- Sheva, Israel. 4Department of Otorhinolaryngology Head and Neck, Institute of expansion phase 1b trial. Lancet Oncol. 20,570–580 (2019). Otorhinolaryngology, Tianjin First Central Hospital, Tianjin, China. 5School of 14. Chou, T. C. & Talalay, P. Quantitative analysis of dose-effect relationships: the Medicine, Tianjin Tianshi College, Tianyuan University, Tianjin, China combined effects of multiple drugs or enzyme inhibitors. Adv. Enzym. Regul 22,27–55 (1984). Author contributions 15. Pilie, P. G., Tang, C., Mills, G. B. & Yap, T. A. State-of-the-art strategies for S.Z., X.P., and X.L. performed the experiments. H.L., B.Z., and Y.L. analyzed the data. M. targeting the DNA damage response in cancer. Nat. Rev. Clin. Oncol. 16, E. and W.W. provided technical assistance. S.Z. and R.W. wrote the main manuscript. 81–104 (2019). R.W. and D.K. revised the manuscript. S.Z., Y.Z., and D.K. designed the experiments. 16. Carlsson, S. K., Brothers, S. P. & Wahlestedt, C. Emerging treatment strategies for All authors reviewed and approved the final version of the manuscript. glioblastoma multiforme. EMBO Mol. Med. 6,1359–1370 (2014). 17. Rasmussen, R. D. et al. BRCA1-regulated RRM2 expression protects glio- Data availability blastoma cells from endogenous replication stress and promotes tumor- The datasets used and/or analyzed during the current study are available from igenicity. Nat. Commun. 7, 13398 (2016). the corresponding author on reasonable request. 18. Netland, I. A. et al. Treatment with the PI3K inhibitor buparlisib (NVP-BKM120) suppresses the growth of established patient-derived GBM xenografts and prolongs survival in nude rats. J. Neurooncol 129,57–66 (2016). Ethics statement 19. Wang, D. et al. Effective use of PI3K inhibitor BKM120 and PARP inhibitor All animal experiments were performed in accordance with the National Olaparib to treat PIK3CA mutant ovarian cancer. Oncotarget 7, Institutes of Health Guide for the Care and Use of Laboratory Animals. 13153–13166 (2015). 20. Matulonis,U.A.etal.PhaseIdoseescalation study of the PI3kinase pathway fl Con ict of interest inhibitor BKM120 and the oral poly (ADP ribose) polymerase (PARP) inhibitor The authors declare no competing interests. olaparib for the treatment of high-grade serous ovarian and breast cancer. Ann. Oncol. 28,512–518 (2017). 21. Ibrahim, Y. H. et al. PI3K inhibition impairs BRCA1/2 expression and sensitizes Publisher’s note BRCA-proficient triple-negative breast cancer to PARP inhibition. Cancer Disco. Springer Nature remains neutral with regard to jurisdictional claims in 2, 1036–1047 (2012). published maps and institutional affiliations. 22. 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