Augmented antitumor activity by olaparib plus AZD1775 in gastric cancer through disrupting DNA damage repair pathways and DNA damage checkpoint

Gastric cancer (GC) is one of the most common malignancies and a leading cause of cancer-related mortality in China [1]. Although emerging targeted strategies have brought new hope to antitumor therapy, options for advanced GC with high heterogeneity are still few, only three drugs (trastuzumab, ramucirumab and apatinib) have been currently approved, and the prognosis of advanced GC remains poor. Hence, development of novel strategies against advanced GC is urgently needed.

Poly ADP-ribose polymerase (PARP) inhibitors that competitively combine and trap PARP to disrupt (SSB) single-strand DNA breaks repairs and elicit anticancer activity emerge as a promising strategy for GC [2‐4]. However, PARP inhibitors alone exert limited efficacy in the treatment of cancers and how to optimize PARP inhibitors’ eligible populations and effectiveness remain poorly understood. Of interest, SSB can be converted into double-strand DNA breaks (DSB), which results in treatment failure of targeting PARP when homologous recombination (HR) is functional [2, 3]. Thus, defects in HR has been identified as a predictor for PARP inhibitors’ sensitivity. For instance, PARP inhibitors olaparib and rucaparib have been approved to treat BRCA-defective ovarian or prostate cancer patients [5] while GC patients harboring low-ATM gains greater survival benefit than high-ATM patients when treated with olaparib plus paclitaxel [4]. Cancers deficient in alternative HR-related factors like RAD51, 53BP1, ARID1A and CCDC6 are also proved sensitive to PARP inhibitors [3, 6, 7]. Based on these, compromising HR functions has been proposed to improve PARP inhibitors’ efficacy against cancers and even expand uses of PARP inhibitors to a greater population with functional HR [8‐13]. However, whether HR deficiency inducers enhance responses of GC to PARP inhibitors and its underlying mechanisms remain uninvestigated, which largely restricts the use of PARP inhibitors.

WEE1 kinase is a gatekeeper of the DNA damage checkpoint (a.k.a. G₂/M checkpoint) that allows DNA repair before mitotic entry [14]. As validated in preclinical models, WEE1 suppression is an emerging strategy against GC [15]. Of note, targeting WEE1 results in HR defects [16‐19], suggesting WEE1 blockade as a promising option for PARP inhibitor-contained combination strategies. However, apart from an ongoing phase Ib study addressing WEE1 inhibitor AZD1775 combined with olaparib against refractory solid tumors (www.​clinicaltrials.​gov; NCT02511795), therapeutic potentials of PARP/WEE1 dual blockade and its effect on HR impairment against cancer remain to be revealed. Moreover, the widely-used WEE1 inhibitor AZD1775 also targets Polo-like kinase 1 (PLK1) that has been reported to impact on PARP inhibitor’s efficacy [20, 21]. Nevertheless, whether PLK1 inhibition by AZD1775 play a critical role in effectiveness of AZD1775-PARP inhibitor combination is elusive.

In this work, we investigated the therapeutic potential and underlying mechanisms of a PARP inhibitor olaparib plus a WEE1/PLK1 dual inhibitor AZD1775 in GC cell lines and preclinical models. Our study sheds light upon the improvement of current PARP targeted therapy and provides evidence for further clinical investigation.

Despite that targeted therapy has unveiled a new era to cancer treatment, current strategies for advanced GC is insufficient to remarkably improve patients’ prognosis. Hence, identifying potential novel targets and developing new combination strategies are critical to benefit new subgroups and optimize effectiveness of current therapy against GC. PARP inhibitors targeting DDR pathways exhibit potent anticancer activity in preclinical models and clinical studies of GC, especially in those with low ATM or RAD51 expression [4, 31, 32]. Due to a close association between efficacy of PARP inhibitors and HR impairment, more and more combination strategies with HR deficiency inducers have been developed against many cancers [8‐13]. However, dual inhibition of PARP and HR functions has not yet been investigated in GC.

Of interest, targeting WEE1, an alternative promising strategy against GC [15], can impair HR functions and is proposed to be a sensitizer of PARP inhibitors [14]. For the first time, our data show that by inducing growth inhibition and apoptosis, combination of the PARP inhibitor olaparib with the WEE1/PLK1 dual inhibitor AZD1775 exerted evidently enhanced antitumor effects in GC cell lines and xenografts than either olaparib or AZD1775 alone (Figs. 1, 2 and 5). Apart from efficacy, safety and tolerability are also key issues in the treatment of GC, especially for combination strategies. Olaparib has been approved to treat BRCA-mutated ovarian cancer at the dose of 400 mg twice a day [33] and proved well tolerated for patients with advanced GC at the dose of 100 mg twice daily in clinical trials [4, 34]. Given the clinical data and olaparib’s daily dose of 25-100 mg/kg extensively-used in mice experiments [13, 32, 35‐37], 25 mg/kg equal to about 140-150 mg in human with 70 kg of body weight [38, 39] was applied in our GC mice studies. As for AZD1775, the human maximum tolerated dose of 225 mg twice daily [40, 41] equates to a rough 79 mg/kg daily dose for mice. Based on previous mice experiments [42‐46], daily oral administration of 30 mg/kg AZD1775 for 3 weeks were used in our work. Consistently, our GC xenograft models had no weight loss after exposure to olaparib (25 mg/kg/d) with/without AZD1775 (30 mg/kg/d) (Fig. 5a), lower than their clinically achievable levels, suggesting safety and tolerability of this combination strategy in GC.

WEE1 repression has been reported to compromise HR functions marked by reduced RAD51 and 53BP1 along with subsequent accumulated γH2AX [16‐19]. Consistently, our findings manifest a downregulation of RAD51 and 53BP1 and an upregulation of γH2AX in GC cell lines and xenografts treated with AZD1775 (Fig. 3a and 5c). RAD51 and 53BP1 was also decreased by olaparib in the presence of AZD1775 compared to olaparib alone, further confirming that AZD1775 potentiated olaparib’s efficiency in GC via impairing HR. Alternative HR-related proteins, such as MRE11, NBS1 and ATM, have been also reportedly lowered by modulation that disrupts HR to improve antitumor efficacy of PARP inhibitors [11, 12]. Resembling RAD51 and 53BP1, responses of these factors to olaparib were replenished by AZD1775 administration (Fig. 3a and 5c), emphasizing that HR impairment was indispensable for the augmented anticancer activity of AZD1775-olaparib combination. As reported, PARP inhibitors primarily blocked repairs of SSB to exert anticancer activity; however, SSB converted into DSB, which could be repaired by HR feedback reactions to impede PARP inhibitors’ responses in HR-functional populations [2, 3]. As an improvement, our data show that AZD1775 in combined therapy caused HR impairment and prevented DSB from repairs to augment DNA damage-mediated cytotoxicity against GC. WEE1/PLK1 blockade provided a HR-defective context, which served as a replenishment to PARP inhibitor functions and gave a rationale for the enhanced antitumor efficacy of the AZD1775-olaparib combination scheme (Fig. 6). Of note, HR dysfunctions that provide an opportunity of optimizing PARP inhibitors’ antitumor efficiency are prevalently evaluated by defective total HR-mediated DDR protein levels beyond which phosphorylation status or immunofluorescent foci of these proteins as well as HR function assays are widely-used methods to monitor HR activity [12, 13, 17, 25, 47]. Thus, other HR function experiments warrant HR impairment’s contribution to enhanced efficacy of AZD1775 plus olaparib against GC in future studies. Besides, PARP trapping serves as key actions for PARP inhibitors in the treatment of cancer [2, 23] and thus we also assessed effects of AZD1775-olaparib combination on it using methods as previously described [23]. Additional file 2: Figure S2b demonstrates olaparib-mediated trapping of PARP to chromatin marked by an increased ratio of trapped PARP1 to total PARP1, however, AZD1775 had no PARP trapping ability and failed to augment olaparib-induced PARP trapping in GC cells. Similarly, PARP catalytic activity, another pivotal factor of PARP inhibitors’ cytotoxicity [23], was inhibited by olaparib indicated by a robust reduction of PARs compared to controls whereas AZD1775 almost had no impact in GC cells (Additional file 2: Figure S2b). Taken together, PARP trapping to chromatin and PARP catalytic inhibition were responsible for PARP inhibitors’ efficacy but dispensable for augmented cytotoxicity of olaparib-AZD1775 combination in GC. Since SSB accumulations followed by replication stress enhancement are a major cause behind single-agent antitumor activity of AZD1775 [23, 46], we also investigated AZD1775-olaparib combination’s impacts on replication stress-associated ATR/Chk1 cascade. Additional file 2: Figure S2a revealed AZD1775-enhanced ATR/PRA32/Chk1 phosphorylation and similar/a bit larger effects when combined with olaparib (phosphorylation sites of PRA32 or Chk1 used are ATR/WEE1 dependent [23, 48‐50]). Thus, AZD1775 induced excessive SSB accumulations and replication stress in our GC cells consistent with previous publications [23, 49, 51], which may facilitate SSB’s conversion to DSB and ensuing AZD1775-induced HR defect in synthetic lethality with olaparib.

Lethality of disrupting DNA damage repairs can be mitigated by functional DNA damage checkpoint (a.k.a. G₂/M checkpoint) that allows DNA repairs before mitotic entry and keeps cancer cells with unrepaired DNA lesions from death [14, 27]. WEE1 inhibitors primarily target DNA damage checkpoint [14, 27], enabling DNA damage checkpoint inactivation to augment lethality of HR deficiency in the context of dual PARP/WEE1 inhibition. As marked by an alteration in G₂/M checkpoint-related proteins (upregulated cyclinB1 and CDC2 phosphorylation; Fig. 4a-c), olaparib-induced G₂/M arrest resulted from an activation of G₂/M checkpoint [18, 28]. WEE1 inhibition has been reported to improve efficacy of G₂ arrest inducers through G₂/M checkpoint inactivation [18, 52], which is dependent on WEE1 blockade’s effects on CDC2 de-phosphorylation [18, 29, 53]. In our GC cells, AZD1775 abrogated olaparib-activated G₂/M checkpoint through CDC2 de-phosphorylation and AZD1775-olaparib combination forced mitotic progression with unrepaired DNA lesions (Fig. 4c-e), called mitotic DNA damage that is responsible for an potentiated anticancer efficiency of AZD1775-combined strategies [18, 19, 29, 52‐54]. Beyond mitotic DNA damage, well-described AZD1775-mediated prolonged mitosis also contributes to pHH3 increment and apoptosis seen in our work (Fig. 2 and 4d). As anticipated, mitosis exit after nocodazole release (marked by a gradual decline in cyclinB1) [55, 56] could be impeded by AZD1775 in our models, which relied on AZD1775-mediated CDC2 de-phosphorylation/activation (Additional file 2: Figure S2c). These data suggest presence of AZD1775-mediated prolonged mitosis in GC cells [55, 57]. In the context of forced mitotic progression and prolonged mitosis induced by AZD1775, AZD1775-olaparib combination ensued to accumulate DNA damage and increase apoptosis (Figs. 2, 5 and 6). Consistent with our data, DNA damage-associated apoptosis can be mediated by HR defect and DNA damage checkpoint inactivation [10‐12, 14, 26, 30]. Taken together, olaparib plus AZD1775 augmented DNA damage-mediated growth inhibition and apoptotic cell death by disrupting HR functions and the DNA damage checkpoint in GC.

Recently, dual targeting of PLK1 and WEE1 has been reported to elicit AZD1775’s single agent cytotoxicity in lung cancer [20]. However, molecular mechanisms underlying AZD1775-decresed exprexssion of the new target PLK1 as observed in our study (Fig. 1a, e and 5c) remain uninvestigated. Intriguingly, several AZD1775’s functions can be partially explained by PLK1 inhibition. For one thing, AZD1775-reduced PLK1 may contribute to AZD1775-induced DNA damage and apoptosis still presented in WEE1 CRISPR-Cas9 knockout cancer cells [20], in line with reliance of AZD1775’s cytotoxicity on targeting PLK1 and WEE1 in our GC cells (Figs. 1 and 2). We also for the first time demonstrated that PLK1 knockdown lowered HR-related proteins as WEE1 knockdown and AZD1775 (Fig. 3), suggesting that PLK1 inhibition-mediated HR defect potentiated DNA damage-associated anticancer efficacy of olaparib plus AZD1775 against GC, resembling WEE1 blockade. For another, the serine/threonine kinase PLK1, governs several mitotic steps, especially mitotic exit and entry into mitosis, keeping cancer cells from genome instability [58]. The spindle assembly checkpoint activation that often delays mitotic exit can be triggered by potent PLK1 inhibitors like AZD1775 and WEE1 knockdown [55, 57, 58], which gives a rationale for AZD1775-prolonged mitosis (Additional file 2: Figure S2c). Of note, PLK1 blockade can directly inhibit WEE1’s phosphorylation and ensuing proteasomal degradation [14] to yield opposite effects on CDC2 activity and DNA damage checkpoint to WEE1 knockdown and AZD1775 (Fig. 4). Moreover, suppression of olaparib-induced PLK1 upregulation can optimize olaparib’s anticancer efficacy in p53-mutated prostate cancer rather than those with p53 wild type [21]. AZD1775 plus olaparib had similar effects in our p53 wild type GC cells (MKN45 and AGS; Fig. 1) despite unknown mechanisms underlying the discrepancy. Thus, whether dual PLK1/WEE1 inhibition elicits anticancer efficacy superior to WEE1 blockade, especially in the presence of olaparib in GC, merits further investigations.

By disrupting DDR pathways and the DNA damage checkpoint to increase DNA damage and apoptosis, WEE1/PLK1 dual inhibitors augmented PARP inhibitors’ antitumor activity in GC cells and xenografts. Hence, the combination scheme with WEE1/PLK1 dual inhibitors may potentially optimize PARP inhibitor’s effect against GC and extend PARP inhibitor’s accessibility from HR-defective to a larger range of HR-proficient patients, which will be helpful for future clinical settings on cancer targeted therapy.