Supplementary Table S5 shows Cell proliferation IC50 and maximum inhibition (Emax) in parental and ERY537S (heterozygous) after different ER ligands treatment.

Supplementary Table S3 shows The effect of ZN-c5 and tamoxifen on E2-induced genes

Supplementary Figure S1 shows ZN-c5 is a novel SERD that inhibits growth of ER + tumors in vitro and in vivo A. ERα degradation curve after ZN-c5 treatment and DC50 (concentration at 50% degradation) determination for Figure 1B top; B. Top: western blot analysis of ERα degradation at 24 hours after fulvestrant treatment in MCF-7 cells at indicated concentration; Bottom: ERα degradation curve and DC50 determination for fulvestrant; C. Tumor growth curve of MCF-7 xenograft model. Mice bearing MCF-7 tumor cells were dosed orally once a day for 26 days. D&E, body weight changes after ZN-c5 or fulvestrant treatment.

PURPOSE Molecular response (MR) based on circulating tumor DNA (ctDNA) is emerging as a promising early biomarker of treatment efficacy in solid tumors; however, its clinical utility in high-grade serous ovarian cancer (HGSOC) remains to be established. This study evaluates the potential predictive value of ctDNA-based MR in with patients HGSOC treated with the WEE1 inhibitor azenosertib. METHODS Plasma cell-free DNA was collected at baseline and after the first and/or second cycle of treatment from 123 patients with recurrent HGSOC enrolled in clinical trials of azenosertib (N = 123). Using the set samples evaluable by high throughput DNA sequencing, MR was defined as a reduction of TP53 variant allelic fraction >50% at the earliest evaluable on-treatment time point. MR was compared with radiographic response per Response Evaluation Criteria in Solid Tumors v1.1, time to progression (TTP), and CA-125 dynamics. RESULTS Molecular responders showed a higher objective response rate ( P = .012, odds ratio = 0.26), greater tumor shrinkage ( P = 5.1e-5, Kruskal-Wallis), and a significantly longer TTP than nonresponders (TTP median 5.49 months v 2.69 months, P = 3.8e-5, hazard ratios [HR] = 0.43). MR was able to identify patients with prolonged survival among those having stable disease at the first radiographic assessment ( P = 3.8e-3, HR = 0.44), and their MR preceded the best overall response by several weeks. Accounting for matching collection time points, MR was evaluable in more patients than CA-125, 88% (102 of 116) versus 71% (85 of 116), respectively, leading to similar predictive value (HR = 0.41 v 0.45, TTP evaluation). CONCLUSION Our findings support the validity and potential clinical utility of ctDNA-based MR as a minimally invasive, rapid, and reliable early surrogate end point in HGSOC.

Supplementary Figure S5 shows ZN-c5 activity in ERY537S tumors. A. Key ER downstream targets expression by qPCR in WT or ERY537S heterozygous cells. B. ER downstream target GREB expression by qPCR in WT or ERY537S heterozygous cells after treatment with 0.1nM E2, 100nM ZN-c5 or 100nM fulvestrant. C. Western blot analysis of ER and PR protein after different concentrations of ZN-c5 treatment for the indicated time in MCF7 ERY537S heterozygous cells. ER or PR protein change (%) relative to non-treatment control after correction by GAPDH. D. WHIM20 tumor bearing mice were dosed with ZN-c5 or fulvestrant at PR protein level in WHIM20 tumor samples harvested at the end of an efficacy study. ZN-c5 was dosed at 40mg/kg, fulvestrant was dosed at 5mg/dose. E. ER expression in WHIM20 tumors by immunohistochemistry (IHC) at the end of efficacy study

Antibody-drug conjugates (ADCs) have transformed targeted cancer therapy, yet strategies to overcome resistance and enhance efficacy remain needed. Since ADCs exert anti-tumor effects via DNA damage or mitotic disruption, combining them with WEE1 inhibitors represents a rational approach. We investigated the selective WEE1 inhibitor azenosertib in combination with ADCs carrying topoisomerase I inhibitor (TOP1i) or microtubule inhibitor (MTI) payloads. Azenosertib enhanced the activity of free TOP1i agents and TOP1i-based ADCs (trastuzumab deruxtecan [T-DXd] and sacituzumab govitecan), increasing DNA damage and apoptosis, and extended the duration of response while overcoming T-DXd resistance in patient-derived xenografts. Synergistic effects were also observed with MTI agents and MTI-based ADCs (mirvetuximab soravtansine, tisotumab vedotin, and enfortumab vedotin), associated with exacerbated mitotic defects and prolonged mitotic arrest. All combinations enhanced efficacy and were well tolerated in vivo. These findings position azenosertib as a broadly applicable enhancer of cytotoxic-payload ADCs, offering a promising strategy for patients with advanced solid tumors.

Supplementary Table S4 shows The effect of ZN-c5 and tamoxifen on E2-suppressed genes

Supplementary Table S2 shows ZN-c5 human nuclear receptor profiling and estrogen receptor binding affinity

Supplementary Table S1 shows the Representativeness of Study Participants

Supplementary Figure S3 demonstrates ZN-c5 protects bone loss in ovariectomized mice A. Progesterone level in serum was measured after Ovariectomy (OVX) or sham surgery to confirm the success of ovariectomy. B and C. BMD in femur and tibia were monitored by InAlyzer system overtime after drug treatment. D. Micro-CT analysis of tibia microarchitecture after at the end of study (12 weeks of drug treatment). E & F. BMD measured by micro-CT in femur and tibia. G. The ratio of uterine wet weight to body weight post-mortem. Statistical significance was evaluated by 2-Way ANOVA, *P < 0.05 compared with sham vehicle. #P < 0.05, ##P < 0.01, compared with OVX-vehicle. Sham, surgery control; OVX, Bilateral ovariectomy

Supplementary Figure S2 shows ZN-c5 demonstrates minor agonist effect on uterine tissue A. Uterine wet weight change in juvenile rats treated with different doses of ZN-c5. B. H&E staining on uterine tissues after ZN-c5 treatment. Images were digitally scanned at 20X magnification

Supplementary Figure S4 shows Combination of ZN-c5 and palbociclib further arrests the cells at G1 phase A. Relative ER level to DMSO after normalized to β-actin control for Figure 4B. B. MCF-7 cells were treated with 500nM of palbociclib for indicated time, cyclin D1 and p-Rb expression were examined by western blot. C. MCF-7 cells were treated with 100nM ZN-c5 or fulvestrant, 500nM palbociclib, or combination of palbociclib with ZN-c5 or fulvestrant, for 24 hours. Western blot analysis of cyclin D1, pRb, ERα and PR were performed. D. cell cycle analysis of MCF-7 cells treated with ZN-c5 or palbociclib for 48 hours.

Supplementary Table S6 shows ER and PR IHC staining average H-score

Cyclin-E1 protein overexpression, including through CCNE1 gene amplification, is recognized as a poor prognostic factor in high-grade serous ovarian cancer (HGSOC) and is a promising predictive marker for investigational therapies targeting cell-cycle checkpoints. However, the demonstration of its clinical utility remains elusive due to inconsistent definitions of protein overexpression and gene amplification. This study characterizes Cyclin-E1 overexpression and CCNE1 amplification prevalence and prognostic value in HGSOC using both original and public clinical cohorts. Fifty-nine percent of tumors overexpressed Cyclin-E1, more than half of which had no evidence of CCNE1 gene amplification. The prevalence of CCNE1 amplification varied across studies and was higher in interventional studies. Patients with Cyclin-E1 positive tumors had poorer outcomes after adjuvant therapy. Platinum-based chemotherapy increased Cyclin-E1 expression. These patients were less likely to benefit from PARP inhibitors (75% are BRCA-wildtype) or mirvetuximab-soravtansine (67% were not FRα-high), highlighting a distinct patient population in need of novel therapies.

Combination of azenosertib with KRASG12C inhibitors reduces tumor cell growth and induces DNA damage and apoptosis in vitro in 3D. A, Seven-day combination treatment dose matrices in KRASG12C NSCLC cell lines cultured as 3D spheroids. Cell viability was measured by 3D CellTiter-Glo and analyzed using the SynergyFinder tool. The Loewe additivity model is depicted in which scores ≥10 are synergistic. Doses are expressed in μmol/L. Cell lines are arranged from most sensitive to least sensitive to KRASG12C monotherapy (left to right). B, Longitudinal cell growth analysis of NCI-H23 spheroids treated with DMSO, 125 nmol/L azenosertib, 5 nmol/L sotorasib, or 5 nmol/L adagrasib for 7 days. The spheroid area was imaged and calculated every 8 hours using an Incucyte instrument. P < 0.0001 for all comparisons with vehicle; P < 0.0001 for combination compared with monotherapies. C, Top, Western blot of protein expression from NCI-H23 spheroids treated with DMSO, 260 nmol/L azenosertib, 38 nmol/L sotorasib, or 20 nmol/L adagrasib for 24 hours. Bottom, Quantification of γH2AX and c-Casp-3/7 protein levels in response to treatment. Results are expressed as fold change relative to DMSO control. DDR, DNA damage response. KRASi, KRAS inhibitor.

Genomic instability and accumulation of DNA damage are hallmarks of tumor development and progression. To ensure the maintenance of genomic integrity, cells rely on a coordinated DNA damage response network that regulates cell-cycle progression, including activation of WEE1-dependent cell cycle checkpoints. If DNA damage occurs during replication, the WEE1 checkpoint is activated, thereby preventing the progression of the cell cycle. This allows damaged DNA to be repaired before cells enter mitosis, or if the damage is too extensive, induction of apoptosis. These observations have made WEE1 a promising anticancer therapeutic target. Azenosertib (ZN-c3) is a novel, selective, and orally bioavailable WEE1 inhibitor. The antiproliferative activity of azenosertib on cancer cell lines is consistent with a WEE1-dependent mechanism of action exemplified by reduction of pY15-CDK1 levels and increases in DNA damage markers. Azenosertib further exacerbates the effect of replicative stress and DNA damage by allowing cancer cells to prematurely enter mitosis, leading to mitotic catastrophe and apoptosis. Azenosertib has optimized pharmacokinetic and pharmacodynamic properties, yielding robust tumor growth inhibition in a broad range of tumor models, and is highly effective at delaying the duration of tumor regrowth after cessation of treatment. We have explored various dosing schedules in preclinical efficacy models for azenosertib that preserve antitumor activity with minimal toxicity. Phase I studies with azenosertib as monotherapy have shown preliminary clinical activity in patients with advanced solid tumors. The data presented herein support further studies of azenosertib monotherapy across multiple solid tumor indications.

Treatment of an NSCLC model with azenosertib + sotorasib results in reduced proliferation and minor histological changes in vivo.

Treatment of an NSCLC CDX model with azenosertib + sotorasib results in tumor regressions and increased DNA damage and apoptosis in vivo. A, Mean tumor volume ± SEM of subcutaneous NCI-H2122 xenografts in NOD/SCID mice treated for 25 days (n = 9/group). P < 0.0001 for all comparisons with vehicle; P < 0.0001 for combination compared with monotherapies. B, Percent change in tumor volume (ΔTV) of individual mice on day 25 of treatment. Values below 0 indicate regression. C, Mean percent change in body weight relative to day 0 (ΔBW) ± SEM. NCI-H2122 was noted to be cachexic in NOD/SCID mice (seen by minor weight loss in the vehicle group), but treatments were well tolerated and did not exacerbate weight loss relative to vehicle. Red dashed line indicates −15% cutoff in weight change. D, Top, Western blots of protein expression from NCI-H2122 tumors treated with one dose of the indicated compounds. Bottom, Quantification of γH2AX and c-Casp-3/7 protein levels in response to treatment. Results are expressed as fold change relative to DMSO control. DDR, DNA damage response.

Treatment of an NSCLC model with azenosertib + adagrasib results in biomarker changes in vitro and in vivo.

Background Phagocytic clearance by macrophages represents a critical immune surveillance mechanism in cancer liver metastasis. Neutrophils, the most abundant immune cells encountered by cancer cells in circulation, play key roles in metastasis through neutrophil extracellular traps (NETs). Although NETs promote macrophage phagocytosis during infection, whether they regulate phagocytosis during cancer metastasis is unknown. The present study aimed to explore the roles of NETs in regulating macrophage phagocytosis during the seeding process of liver metastasis and the mechanisms underlying the roles.Methods A lipopolysaccharide-induced NET model was applied to study the role of NETs on colorectal cancer (CRC) liver metastasis. The neutrophils isolated from human peripheral blood were stimulated with PMA to release NETs, which were collected and added to the cultures of different CRC cell lines for in vitro studies. Macrophage phagocytosis was assessed with flow cytometry in vitro and in vivo. RNA-seq and microRNA array analyses were performed to identify key pathways regulated by NETs and downstream key molecules. The macrophage phenotypes were evaluated using immunohistochemistry, flow cytometry, and cytokine and chemokine arrays.Results NETs promote macrophage phagocytosis both in vitro and in vivo. Neutrophil elastase (NE), which was able to inactivate the canonical signal of protease-activated receptor 2 (PAR2), downregulated the phagocytotic checkpoint CD24. Notably, PAR2 deficiency imitated the effect of NETs on phagocytosis and CD24. Mechanistic studies indicated that inhibiting PAR2 expression upregulated miR-34a and miR-146a and downregulated CD24 in cancer cells. In addition, PAR2 depletion enhanced the recruitment and M1 polarization of macrophages by upregulating CSF-1 and CXCL1. The correlation of NETs/NE and CD24 was corroborated using human CRC specimens. Furthermore, PAR2 blockade combined with an anti-EGFR antibody (cetuximab (CTX)) synergistically enhanced the phagocytic ability of macrophages and suppressed liver metastasis in vivo.Conclusions NET-derived elastase inactivated PAR2 canonical signaling and promoted phagocytosis by downregulating CD24, which functions as a phagocytotic checkpoint in CRC liver metastasis. Thus, PAR2 inhibitors combined with CTX may serve as a novel therapeutic strategy against advanced CRC.