Elevated expression of WEE1 and CHK1 in CRPC and NEPC cells and tumor samples.
To assess the potential therapeutic value of targeting WEE1 and CHK1 in prostate cancer, we first evaluated WEE1 and CHK1 expression in a panel of prostate cancer cell lines with different genetic backgrounds. As shown in Fig. 1A, the CRPC cell lines (C4-2B, VCaP, 22Rv1, and DU145) exhibited enhanced WEE1 and CHK1 expression compared to those in the androgen-sensitive cell lines LNCaP and LAPC4, and the immortalized normal human prostate epithelial cell line RWPE1 (LAPC4 depicted in Supplemental Fig. S1A). The elevated expression of WEE1 and CHK1 in LNCaP-derived C4-2B CRPC cells relative to parental LNCaP cells indicates that the upregulation of these two genes may contribute to castration resistance. The expression of WEE1 and CHK1 was also elevated in NCI-H660 (H660), an authentic NEPC cell line derived from a clinical NEPC patient tumor. Their expression in two other NEPC cell lines, PC3 and LASCPC-01 (an NEPC cell line derived from benign human prostate tissue through transformation with the MYCN and myristoylated AKT1 oncogenes), was similar to that of LNCaP cells, but higher than that in normal RWPE1 cells. WEE1 protein expression in these cells significantly correlated with that of CHK1 (Pearson’s r = 0.70, p = 0.02, n = 3 Fig. 1B) and p-Y15-CDK1 (Fig. S1B), an indicator of the activated G2/M checkpoint, suggesting that these CRPC and NEPC cells may rely on WEE1- and CHK1-mediated G2/M control for survival, and thereby could be sensitive to agents targeting this pathway.
To assess the significance of WEE1 and CHK1 gene expression in human prostate cancer patients, the relationship between CHK1 and WEE1 mRNA and the pathological features and survival of patients with prostate adenocarcinoma was analyzed using data from The Cancer Genome Atlas (TCGA-PRAD). As shown in Fig. 1C, significantly higher proportions of samples at more advanced stages were observed in the upper quartile than in the lower quartile of CHK1 expression (p = 10E-7.26 for T staging, p = 0.039 for N staging, Supplemental Table S1). Similar results were observed between the WEE1 expression level and pathological T&N staging (p = 10E-3.41 for T staging, p = 10E-4.16 for N staging, Supplemental Table S1). Analysis of overall survival in patients with high or low CHK1 expression indicated that patients with CHK1 expression levels lower than the median achieved a significantly higher probability of survival (p = 0.0103) with a median survival time of approximately 31 months, compared to those expressing CHK1 higher than the median (Fig. 1D). A similar trend was observed between WEE1 expression and survival status, although the difference was not statistically significant (p = 0.4132) (Fig. 1D). The prognostic values of CHK1 and WEE1 were further explored as the survival status became more time-dependent as the disease progressed, especially among subjects with mCRPC. In line with the significant prognostic value of CHK1 as suggested by the Kaplan-Meier (K-M) curves, its power as a prognostic indicator was confirmed by the time-dependent AUC throughout the first 24 months (AUC above 0.6, Fig. 1E). On the other hand, the low AUC values of WEE1 in the first 12 months also matched the insignificant prognostic value reflected by the K-M curves. However, the AUC value of WEE1 went above 0.6 after two years, suggesting an increased importance of WEE1 in contrast to CHK1 at later stages of disease progression (Fig. 1E, Supplemental Table S2). Interestingly, compared to prostate adenocarcinoma, CHK1 mRNA was markedly upregulated in NEPC tumors (p = 7.299E-4), while that of WEE1 was not (p = 0.398) (Fig. 1F)., The expression of CHK1 mRNA was positively correlated with the expression of WEE1 mRNA in NEPC (Spearman’s r = 0.6, p = 4.65E-6), but not in prostate adenocarcinoma (Spearman’s r = -0.13, p = 4.944E-3) (Fig. 1G). Overall, these data provide a rationale for targeting WEE1 and CHK1 in advanced stages of prostate cancer, with CHK1 being more strongly indicated.
Effects of WEE1 or CHK1 inhibition on the survival of CRPC and NEPC cells.
Increased WEE1 and CHK1 expression in CRPC and NEPC cells suggests that these cells may depend on the G2/M checkpoint for survival, and may be sensitive to the inhibition of these two kinases. The WEE1 inhibitor AZD1775 and the CHK1 inhibitor SRA737 were selected to examine this possibility. Both inhibitors are in phase I/II clinical trials for various cancers [21–23]. AZD1775 has been evaluated in clinical trials for solid tumors, including mCRPC, but not in trials designed for prostate cancer. SRA737, on the other hand, has not been evaluated in the context of prostate cancer. This study examined the effects of AZD1775 and SRA737 on the proliferation and viability of CRPC and NEPC cells stably expressing the red fluorescent marker NucLight Red (NR) by real-time live-cell imaging. As shown in Fig. 2A, AZD1775 induced time- and concentration-dependent biphasic responses in DU145 cells, where at lower concentrations (≤ 0.38 µM for DU145), cell proliferation was stimulated as a consequence of increased mitosis due to inhibition of WEE1 while at higher concentrations (> 0.38 µM for DU145) decreased cell viability/proliferation was observed. Similar effects were observed in all other cell lines examined, including 22Rv1, PC3, and C4-2B, and three drug-resistant lines derived from C4-2B, C4-2B-MDVR (enzalutamide-resistant), C4-2B-TaxR (docetaxel-resistant), and C4-2B-AbiR (abiraterone-resistant) [24–26] (Supplemental Fig. S2A). The dose-response curves for AZD1775 at 96 h were plotted, showing a similar pattern of responses with IC50 values between 0.5-1 µM for all cell lines examined except for PC3, which was 2-fold higher (Fig. 2B and Table 1), and the responses did not correlate to WEE1 expression in these cells. The effect of AZD1775 was rapid and plateaued at 48 h and the IC50 values remained constant at 48, 72, 96 h in most cell lines (Fig. 2C). AZD1775 dose-dependently increased mitotic cell numbers, as measured by p-S10-Histone H3 (pHH3) immunofluorescence (IF) staining (Fig. 2D), and resulted in elevated DNA double-strand breaks, as measured by IF staining of p-S139-His H2A.X (γH2AX) foci (Fig. 2E). Accordingly, knockdown of WEE1 by siRNAs significantly reduced cell viability in C4-2B and H660 cells as compared to the control cells transfected with non-targeting siRNA (si-NT) (Fig. 2F). The analysis of SRA737 demonstrated a similar biphasic response in proliferation/viability of all cell lines examined, namely stimulation of proliferation at lower concentrations (≤ 0.78 µM for DU145) while inhibiting at higher concentrations (> 0.78 µM for DU145) (Fig. 2G and supplemental Fig. S2B). However, when dose-response curves for SRA737 were plotted at 96 h, apparent differences in sensitivity to SRA737 were observed between the cell lines. Specifically, 22Rv1 and DU145 were 10- to 20-fold more sensitive to SRA737 than were C4-2B, PC3, and H660 (Fig. 2H and Table 1). Similar to AZD1775, differences in sensitivity to SRA737 did not consistently correlate with CHK1 expression in these cells. Interestingly, the C4-2B drug-resistant cell lines also exhibited approximately 2 to 10-fold greater sensitivity to SRA737 than the parental C4-2B cells (Fig. 2H and Table 1), implying a potential value of SRA737 for therapy-resistant prostate cancer. Additionally, in contrast to AZD1775, SRA737 was more slow-acting, as a time-dependent decrease in IC50 values was observed in all sensitive cell lines. However, this was less apparent in the more resistant C4-2B and PC3 cell lines. A longer incubation time is required to achieve a maximal response to SRA737 (minimally 72 h) (Fig. 2I). Collectively, these data indicate that AZD1775 and SRA737 are effective therapeutic agents for CRPC and NEPC in vitro.
Table 1
IC50 values for AZD1775 and SRA737 in CRPC and NEPC cell lines.
| IC50 (µM)A |
Cell Lines | AZD1775 n = 3 | SRA737 n = 3 |
LNCaPB | 0.26 ± 0.04 | 2.01 ± 0.53 |
22Rv1 | 0.71 ± 0.19 | 1.70 ± 0.33 |
DU145 | 0.70 ± 0.10 | 1.47 ± 0.61 |
C4-2B-MDVR | 0.57 ± 0.23 | 2.21 ± 0.68 |
C4-2B-TaxR | 0.66 ± 0.30 | 1.97 ± 1.15 |
C4-2B-AbiR | 0.77 ± 0.16 | 9.29 ± 5.72 |
C4-2B | 0.58 ± 0.04 | 20.17 ± 5.24 |
PC3 | 1.21 ± 0.69 | 18.60 ± 6.26 |
NCI-H660C | 1.14 ± 0.29 | 23.18 ± 1.08 |
AIC50s were calculated using time-lapse imaging analysis of NR-positive cells treated with increasing concentrations of AZD1775 and SRA737 at 96 h. Data are Mean ± SEM from three independent experiments. |
BIC50s for LNCaP were obtained based on % confluency imaged under phase. |
CIC50s for H660 were obtained from the CCK-8 cell viability assay. |
Synthetic lethal effects of combined WEE1 and CHK1 inhibition in vitro.
The fact that WEE1 and CHK1 act through different pathways to modulate the CDK1/cyclin B complex implies that their inhibition can be combined to enhance abrogation of the G2/M checkpoint. To test this, we examined the antiproliferative effects of AZD1775 and SRA737 alone and in combination in CRPC and NEPC cells using live-cell imaging. As shown in Fig. 3A, the combination of AZD1775 and SRA737 resulted in more significant inhibition of cell proliferation/viability as compared to each drug alone, and the effect was most apparent at non-toxic, low doses of AZD1775 (0.19–0.38 µM) and SRA737 (3.13–6.25 µM) for C4-2B cells. A cooperativity screen was then conducted to further evaluate the interactions between the two drugs. As illustrated in Fig. 3B-C, strong synergy between the two drugs was demonstrated, as indicated by synergy scores obtained from the four synergy analysis models (Fig. 3B, Table 2). The most significant synergy was exemplified in a growth kinetic plot showing the combination of two non-toxic, low doses of AZD1775 (0.38 µM) and SRA737 (3.13 µM) resulted in nearly complete inhibition of C4-2B proliferation (Fig. 3C). Drug interaction analysis demonstrated similar synergistic effects between AZD1775 and SRA737 in 22Rv1, DU145, and PC3 cells (Supplemental Fig. S3A-C and Table 2), with PC3 NEPC cells displaying the greatest synergy score (supplemental Fig. S3C).
Table 2
Synergy scores from AZD1775 and SRA737 cooperativity screen.
| Loewe | Bliss | ZIP | HSA |
Cell lines | Score | p | Score | p | Score | p | Score | p |
22Rv1 | 6.18 | 3.40E-06 | 3.2 | 1.16E-01 | 2.56 | 1.81E-01 | 10.17 | 6.21E-06 |
DU145 | 7.12 | 1.37E-10 | 0.02 | 9.92E-01 | -2.17 | 2.20E-01 | 11.33 | 3.02E-06 |
C4-2B | 8.88 | 1.94E-04 | 20.05 | 7.81E-09 | 17.22 | 8.64E-08 | 15.39 | 7.84E-08 |
C4-2B-MDVR | 9.71 | 7.48E-10 | 7.33 | 1.35E-05 | 6.77 | 1.59E-05 | 12.97 | 1.26E-10 |
PC3 | 18.98 | 9.27E-09 | 32.43 | 1.96E-13 | 31.17 | 8.79E-12 | 25.13 | 4.89E-12 |
LASCPC-01 | 4.62 | 8.91E-07 | 9.78 | 6.88E-08 | 9.06 | 2.05E-06 | 13.97 | 9.46E-32 |
Cell viability (%) was calculated by Incucyte live cell imaging analysis. Drug interactions at day 4 were analyzed by SyngeryFinder Plus. Synergy scores and p values were obtained from four synergy analysis models. Representative data from one of three independent experiments are shown. |
Because the NEPC cell line LASCPC grew in partially suspended clusters, NR-positive tumor spheroids were established for these cells. As shown in Fig. 3D, AZD1775 and SRA737 suppressed the growth of LASCPC tumor spheroids in time- and dose-dependent manners. Their combination resulted in greater retardation of growth kinetics than each drug alone. The cooperativity screen of the AZD1775 and SRA737 combination demonstrated strong synergy, as indicated by their synergistic 3D plot and scores (Fig. 3E-G, Table 2). In a representative spheroid growth plot, the combination of two non-toxic, low doses of AZD1775 (0.38 µM) and SRA737 (1.56 µM) completely eradicated the growth of LASCPC tumor spheroid. Collectively, these data demonstrate a strong synergy of AZD1775 and SRA737 combination in 2D CRPC and NEPC cell cultures and 3D tumor spheroids, suggesting that the combined inhibition of WEE1 and CHK1 may be an effective treatment for advanced prostate cancer.
CHK1 inhibition synergized with WEE1 inhibition by blocking WEE1 inhibitor-induced feedback activation of CHK1 in prostate cancer cells.
In this study, we examined the molecular mechanisms underlying the synergistic effects of AZD1775 and SRA737 on prostate cancer cells. As shown in Fig. 4A, the two drugs at their respective IC50 concentrations had distinct effects on cell cycle and DNA damage markers in DU145 cells. AZD1775 rapidly inhibited p-Y15-CDK1 observed 30 min after treatment initiation, while SRA737 did not affect p-Y15-CDK1 for up to 24 h. Correspondingly, AZD1775 rapidly and potently stimulated mitotic entry (pHH3), which peaked at 3 h, while only a small effect was observed for SRA737. In contrast, both drugs induced time-dependent DNA damage (γH2AX) which peaked at 3 h for AZD1775 and 24 h for SRA737. Interestingly, SRA737 also caused a time-dependent downregulation of CHK1, which was visible after 3 h and progressed over time. Little effect was observed for p-S216-CDC25 or CDC25C at any time point, except at 24 h. These data imply that AZD1775 acts primarily by regulating the cell cycle, whereas SRA737 is a weaker cell cycle regulator and has more complex mechanisms of action, likely involving downregulation of CHK1 and induction of DNA damage. Moreover, both AZD1775 and SRA737 induced p-S345-CHK1 expression, an indicator of CHK1 activation by ATR (Fig. 4A). CHK1 inhibitors are known to induce p-S345-CHK1 through a CHK1 activity-dependent phosphatase cycle, where inhibition of CHK1 causes the accumulation of p-S345-CHK1 [27]. Inhibition of WEE1 by AZD1775 has also been shown to induce p-S345-CHK1 by activating ATR in other cancer cells [28], which may circumvent the effectiveness of WEE1 inhibition. AZD1775-induced p-S345-CHK1 was also observed in other prostate cancer cells, such as H660 and C4-2B, where increased p-S345-CHK1 was accompanied by increased pHH3 and γH3AX (Fig. 4B). This effect was dependent on ATR, because blocking ATR by AZD6738, an ATR inhibitor, significantly attenuated the levels of p-S345-CHK1 induced by AZD1775 and increased mitotic entry (pHH3) in DU145 cells (Fig. 4C). This was an on-target effect of WEE1 inhibition since knockdown of WEE1 also caused a robust increase in p-S345-CHK1, accompanied by increased mitosis (pHH3) and DNA damage (γH3AX) in H660 cells (Fig. 4D). Thus, these data provide a rationale for combining WEE1 inhibition with CHK1 inhibition to achieve synergistic killing of prostate cancer cells.
Our data demonstrated a strong synergy between AZD1775 and SRA737 in all prostate cancer lines regardless of their differential sensitivity to SRA737. To understand the molecular underpinnings of this observation, the effects of both agents on key cell cycle regulators were compared between SRA737-sensitive and -resistant cells. As shown in Fig. 4E, in SRA737-resistant H660 cells, both AZD1775 and SRA737 at their respective IC50 doses induced a robust increase in p-S345-CHK1, possibly contributing to the lack of effect on p-Y15-CDK1. This contrasted with the almost complete blockade of p-Y15-CDK1 in the two SRA737-sensitive LASCPC and LNCaP cell lines. Interestingly, despite the different responses to single drugs. The combination of AZD1775 and SRA737 was equally potent at inhibiting p-Y15-CDK1 in both sensitive and resistant cells, correlating well with the uniformly strong synergy in all cell lines. The blocking of p-Y15-CDK1 marks the activation of CDK1/cyclin B, leading to premature entry into mitosis (pHH3) and accumulation of DNA damage (γH3AX), which could be observed in both sensitive and resistant cells with varying magnitude and kinetics (Fig. 4E). Significant downregulation of CHK1 was also detected in response to each drug and their combination, although this could be the consequence of an altered cell cycle. Collectively, blockade of AZD1775-induced feedback activation of CHK1 by SRA737 is likely the core mechanism driving the synergy between AZD1775 and SRA737 in prostate cancer cells.
Inhibition of WEE1 or CHK1 suppressed tumor growth and metastasis in vivo.
To determine the therapeutic potential of AZD1775, SRA737, and their combination for treating advanced prostate cancer, we evaluated the efficacy in vivo using the castrated TRAMP mouse model, the best-known transgenic mouse model of NEPC. NEPC represents the most advanced stage of lethal prostate cancer, for which no effective therapies are available. TRAMP mice were routinely obtained as [TRAMPxFVB] F1 offspring. TRAMP tumors are characterized by rapid onset and metastatic progression with complete penetrance, and castration leads to the development of castration-resistant neuroendocrine carcinomas with lymph node and distal metastases [29]. We first evaluated the efficacy of AZD1775 and SRA737 as single agents in a 4-week simultaneous treatment regimen. TRAMP mice castrated at 16–17 weeks of age were randomized into three treatment groups: vehicle (n = 8), AZD1775 (60 mg/kg, n = 7), and SRA737 (100 mg/kg, n = 7) (Fig. 5A). All treatments were administered orally on 5 days-on / 2 days-off for four cycles. The mice were euthanized after four weeks. In mice with palpable tumors, tumor size was determined twice a week until the end of the experiment. Owing to the considerable variability in tumor volumes and stages, data from mice with or without palpable tumors were stratified and analyzed separately. In mice with palpable prostate tumors, AZD1775 or SRA737 administered alone not only completely blocked tumor growth but also resulted in tumor regression in both treatment groups (*p = 0.012 for AZD1775; **p = 0.004 for SRA737) (Fig. 5B-C and statistical analysis in Supplemental Table S3). In mice without palpable tumors, the final mean urogenital tract (UGT) weight from AZD1775- or SRA737-treated mice was significantly lower than that of vehicle-treated mice (**p < 0.002) (Fig. 5D and statistical analysis in supplemental Table S4). Correspondingly, the sizes of the UGTs were also smaller in drug-treated mice (Fig. 5E). Each drug was well tolerated because mouse body weight remained constant throughout the treatment (p = 0.352) and no other adverse effects were observed (Fig. 5F and statistical analysis in Supplemental Table S4). Kaplan–Meier survival analysis of mice treated with AZD1775 or SRA737 showed improved overall survival compared to the vehicle group, but the difference was not statistically significant (p = 0.20 with log-rank test) (Fig. 5G).
We demonstrated elevated WEE1 expression and potent cell-killing effects of AZD1775 in NEPC cells and tumor spheroids (Fig. 1), which is consistent with the evidence that WEE1 was previously identified as a potential target for NEPC tumors [6]. To fully exploit the therapeutic potential of WEE1 inhibition, we further examined the effectiveness of AZD1775 in a mouse survival experiment, in which treatment was initiated upon the development of palpable primary prostate tumors and continued until death or humane moribund endpoints. TRAMP male mice were castrated at 15–18 weeks. Upon development of palpable tumors, the mice were randomly assigned to two experimental groups: 1. vehicle (PO, qd, n = 5), 2. AZD1775 (60 mg/kg, PO, qd, n = 6). AZD1775 treatment for up to 12 weeks was well tolerated, with no significant change in body weight (p = 0.818) or apparent gross toxicity (Fig. 5H). Our data demonstrated that AZD1775 improved the overall survival of TRAMP mice compared to vehicle-treated mice, though did not reach statistical significance (p = 0.07 with log-rank test) (Fig. 5I).
In addition to tumor growth, the incidence of metastatic spread was examined visually at necropsy, and metastatic lesions were confirmed histologically. In this mouse model, lymph nodes were the most common sites of metastases, which occurred in 33%-40% of castrated mice at 24–25 weeks. As shown in Table 3, the incidence of lymph node (LN) metastases was lower in the AZD1775- and SRA737-treated groups than in the vehicle group. Metastases in other distal organs (liver, lung, and kidney) were also lower in drug-treated groups. Additionally, the incidence of newly developed primary prostate tumors during treatment was reduced in AZD1775- and SRA737-treated mice (Table 4). Taken together, the inhibition of WEE1 or CHK1 effectively suppresses the growth and metastasis of NEPC prostate tumors, supporting their values as single agents for prostate cancer treatment.
Table 3
Incidence of distant metastases confirmed histologically.
| | Pelvic LN | Renal LN | Inguinal LN | All 3 LNs | Distal Organs |
Treatment | NA | +B | % | pD | +C | % | p | +C | % | p | +C | % | p | +C | % | p |
4-wk treatment | | | | 1 | | | 0.726 | | | 1 | | | 0.726 | | | 0.623 |
Vehicle | 5 | 2 | 40 | | 1 | 20 | | 0 | 0 | | 1 | 20 | | 2 | 40 | |
AZD1775 | 7 | 2 | 29 | 1 | 14 | 1 | 14 | 1 | 14 | 1 | 14 |
SRA737 | 6 | 2 | 33 | 0 | 0 | 1 | 17 | 0 | 0 | 1 | 17 |
2-wk sequential treatment | | | | 0.305 | | | 1 | | | 0.084 | | | 0.648 | | | 0.207 |
Vehicle | 20 | 11 | 55.0 | | 5 | 25.0 | | 4 | 20.0 | | 1 | 5.0 | | 3 | 15.0 | |
AZD1775 | 20 | 11 | 55.0 | 4 | 20.0 | 6 | 30.0 | 3 | 15.0 | 1 | 5.0 |
SRA737 | 23 | 9 | 39.1 | 5 | 21.7 | 1 | 4.3 | 1 | 4.3 | 0 | 0 |
AZD1775 + SRA737 | 25 | 8 | 32.0 | 6 | 24.0 | 2 | 8.0 | 2 | 8.0 | 1 | 4.0 |
ATotal number of mice in each experimental group, including all mice with or without palpable tumors at the beginning of the treatment. The numbers do not include mice that died during the treatment (3 died for vehicle and 1 for SRA737). LN: Lymph node. |
BNumber of mice with positive lymph node metastases in indicated lymph nodes. The presence of metastatic tumor cells in the lymph nodes was confirmed histologically by staining for synaptophysin. |
CNumber of mice with positive metastases in distal organs including urethra, liver, lung, or kidney. The presence of metastases was confirmed visually and histologically by staining for synaptophysin. |
Dp values are obtained from Fisher’s exact test (FET) evaluating the association between developing recurrence and treatment group. Computed FET across all groups (2×3 or 2×4 tables). |
Table 4
Incidence of relapsed and newly occurred primary prostate tumors.
| | Macroscopic recurred prostate tumor | Microscopic recurred prostate tumor |
Treatment | nA | nB | % | pD | nC | % | p |
4-wk treatment | | | | | | | 0.726 |
Vehicle | 5 | 2 | 40.0 | | 1 | 20.0 | |
AZD1775 | 7 | 2 | 28.6 | 1 | 14.3 |
SRA737 | 6 | 2 | 33.3 | 0 | 0 |
2-wk sequential treatment | | | | 0.188 | | | 0.789 |
Vehicle | 14 | 7 | 50.0 | | 2 | 14.3 | |
AZD1775 | 14 | 5 | 35.7 | 1 | 7.14 |
SRA737 | 15 | 2 | 13.3 | 1 | 6.67 |
AZD1775 + SRA737 | 16 | 4 | 25.0 | 3 | 18.8 |
ATotal number of mice in each experimental group including all mice without tumors at the beginning of treatment. The numbers do not include mice that died during the treatment (3 died for vehicle and 1 for SRA737). |
BNumber of mice with palpable (macroscopic) prostate tumors recurred during drug treatment. |
Number of mice with microscopic prostate tumors recurred during drug treatment. The presence of primary tumors was confirmed histologically by H&E and staining for synaptophysin. |
Dp values were obtained from Fisher’s exact test (FET) evaluating the association between developing recurrence and treatment group. Computed FET across all groups (2X3 or 2X4 tables). |
Combined inhibition of WEE1 and CHK1 blocked NEPC tumor progression and metastasis.
The effectiveness and tolerability of AZD1775 and SRA737 as single agents and their strong synergy in prostate cancer cells support their combination for increased efficacy and reduced toxicity. However, in a pilot experiment, a 4-week combination treatment showed signs of severe toxicity, including rapid weight loss and death. Careful optimization of the combination is required to maximize the efficacy and minimize toxicity. Therefore, we evaluated several combination regimens by varying the frequency, duration, and sequence of drug administration, and found that a sequential 2-week schedule was well tolerated. In a large scale study, four groups of randomized castrated TRAMP mice (20–21 weeks) were treated with vehicle (n = 20), AZD1775 (60 mg/kg, PO, 3 days on / 4 days off for 2 cycles, n = 20), SRA737 (100 mg/kg, PO, 2 days on / 5 days off for 2 cycles, n = 23), or AZD1775 (60 mg/kg) + SRA737 (100 mg/kg SRA737)(PO, 3 days AZD1775 followed by 2 days SRA737 / 2 days off for 2 cycles, n = 25) (Fig. 6A). Tumor growth in mice with palpable tumors was determined twice per week until the end of the experiment (Fig. 6C-D). Mice were euthanized after 4 weeks, and necropsy was performed to collect tissues and determine the extent of tumor development and incidence of tumor metastases. Sequential combination treatment was well tolerated. There was no significant difference in the body weight between the groups (p = 0.487) (Fig. 6B). No other gross toxicity was noted, and the internal organs (liver, spleen, and kidney) were normal in size/weight/appearance in all groups of mice (Supplemental Fig. S4A). In mice with palpable prostate tumors, the combination of AZD1775 and SRA737 provided the most potent suppression of tumor growth compared to each drug alone (p < 0.001). SRA737 also showed remarkable single-agent activity in tumor suppression (p < 0.001) compared to AZD1775, which was visible but not significant (p = 0.091) (Fig. 6C and statistical analysis in Supplemental Table S5). Accordingly, SRA737 and its combination also significantly reduced the final tumor weight (p < 0.05) (Fig. 6C-E and statistical analysis in Supplemental Table S6). After the cessation of drug treatment, tumor growth rebounded in the drug-treated groups. Nonetheless, a notable more sustained suppression of tumor growth (lasting until day 18) was observed in the combination-treated group (Fig. 6C). In mice without palpable tumors, the final mean %UGT weight was lower in the SRA737 (0.87 ± 1.98) and the combination groups (1.56 ± 2.49) as compared to vehicle (2.44 ± 3.54) and AZD1775 (2.32 ± 4.06). Still, the difference did not reach statistical significance (p = 0.513) (Fig. 6F). Kaplan–Meier survival analysis of mice treated with AZD1775 or SRA737 showed improved overall survival compared with the vehicle (p < 0.02) (Fig. 5G). The 28-day survival rates were 77% (vehicle), 91% (AZD), 100% (SRA), and 89% (AZD + SRA). Among the mice with tumors, the 28-day survival was 40% (vehicle), 83% (AZD), 100% (SRA), and 70% (AZD + SRA). Survival between these groups was better for all treatment groups than for the vehicle (p = 0.06 for all mice and p = 0.02 for mice with tumors only). Regarding tumor metastasis, the incidence of lymph node metastases at three sites (pelvic, renal, and inguinal LN) was lower in SRA737 and the combination groups than in the vehicle and AZD1775 groups. Metastasis in the distal organs (liver, lung, and kidney) was reduced in all treatment groups compared to that in the vehicle group. Notably, the effect of SRA737 on tumor metastasis was equivalent to or greater than that of the combination (Table 3). Additionally, the incidence of newly occurring primary prostate tumors was lowered in all treatment groups than in the vehicle group, with the most significant effect in the SRA737 group (Table 4). Collectively, these data demonstrated not only the synergistic effects of the AZD1775 and SRA737 combination, but also the effectiveness of SRA737 as a single agent in suppressing NEPC tumor growth and metastasis in castrated TRAMP mice.