Gordon B. Mills

Sequential treatment with PARPi and WEE1i enhances antitumor immune responses in preclinical models of ovarian cancer

The antitumor activity demonstrated by DNA damage response inhibitors (DDRis) can be partially attributed to their capacity to enhance immune responses. However, the toxicity of DDRis to lymphocytes, particularly when a DDRi is combined with other treatments targeting cell cycle checkpoint kinases, indicates a need for the development of different DDRi treatment schedules. Here, we systematically assessed changes to the tumor immune microenvironment (TIME) in response to DDRis across various treatment timelines in ovarian cancer. Using single-cell analysis, we found that the sequential treatment with an inhibitor of poly(ADP-ribose) polymerase (PARPi), followed by an inhibitor of the cell cycle checkpoint kinase WEE1 (WEE1i), resulted in more effective cancer eradication and stronger antitumor immune responses in vivo, compared with mono- and concurrent therapy. Both sequential and concurrent treatment schedules could induce lethal DNA damage and activate the cGAS-STING pathway in cancer cells, but T cell viability was greater under sequential treatment. Proteomic analysis showed that T cells more quickly recovered from DNA damage after DDRi treatment compared with cancer cells. Both immune checkpoint therapy and CAR T cells were more effective when combined with sequential treatment compared with monotherapy treatment in a syngeneic high-grade serous ovarian cancer mouse model and in a treatment-resistant ovarian cancer patient-derived xenograft model. Our study demonstrated that sequential treatment with PARPi and WEE1i spared T cells from severe DNA damage and activated the cGAS-STING pathway in cancer cells, suggesting that antitumor immunity and control of tumor growth can be optimized through changes in treatment schedules.

Proteogenomic insights into early-onset endometrioid endometrial carcinoma: predictors for fertility-sparing therapy response

Endometrial carcinoma remains a public health concern with a growing incidence, particularly in younger women. Preserving fertility is a crucial consideration in the management of early-onset endometrioid endometrial carcinoma (EEEC), particularly in patients under 40 who maintain both reproductive desire and capacity. To illuminate the molecular characteristics of EEEC, we undertook a large-scale multi-omics study of 215 patients with endometrial carcinoma, including 81 with EEEC. We reveal an unexpected association between exposome-related mutational signature and EEEC, characterized by specific CTNNB1 and SIGLEC10 hotspot mutations and disruption of downstream pathways. Interestingly, SIGLEC10

L1CAM is required for early dissemination of fallopian tube carcinoma precursors to the ovary

AbstractMost ovarian high-grade serous carcinomas (HGSC) arise from Serous Tubal Intraepithelial Carcinoma (STIC) lesions in the distal end of the fallopian tube (FT). Formation of STIC lesions from FT secretory cells leads to seeding of the ovarian surface, with rapid tumor dissemination to other abdominal structures thereafter. It remains unclear how nascent malignant cells leave the FT to colonize the ovary. This report provides evidence that the L1 cell adhesion molecule (L1CAM) contributes to the ability of transformed FT secretory cells (FTSEC) to detach from the tube, survive under anchorage-independent conditions, and seed the ovarian surface. L1CAM was highly expressed on the apical cells of STIC lesions and contributed to ovarian colonization by upregulating integrins and fibronectin in malignant cells and activating the AKT and ERK pathways. These changes increased cell survival under ultra-low attachment conditions that mimic transit from the FT to the ovary. To study dissemination to the ovary, we developed a tumor-ovary co-culture model. We showed that L1CAM expression was important for FT cells to invade the ovary as a cohesive group. Our results indicate that in the early stages of HGSC development, transformed FTSECs disseminate from the FT to the ovary in a L1CAM-dependent manner.

Rictor orchestrates β-catenin/FOXO balance by maintaining redox homeostasis during development of ovarian cancer

Rictor/mTORC2 has been demonstrated to have important roles in cancer development and progression in a number of solid and hematologic malignancies. However, little is known about the role of Rictor/mTORC2 in ovarian cancer pathophysiology. Herein, using conditional Rictor knockout mice, we were able to demonstrate that Rictor deletion disrupted glutathione metabolism through AKT/Nrf2 signaling pathway and induced intracellular oxidative stress during the malignant transformation of Kras/Pten-mutant ovarian surface epithelial cells. Elevated reactive oxygen species and activated FOXO3a in Rictor-deleted cells strikingly shifts the functional interaction of β-catenin from TCF to FOXO3a, which strongly inhibits classical Wnt/β-catenin signaling. Our findings emphasize a pivotal role for Rictor in orchestrating crosstalk between the PI3K/AKT and Wnt/β-catenin signaling in the development of ovarian cancer. Illustration of Rictor/mTORC2 in promoting tumor onset by regulating glutathione metabolism and mediating oncogenic signaling.

Peritoneal Spread of Ovarian Cancer Harbors Therapeutic Vulnerabilities Regulated by FOXM1 and EGFR/ERBB2 Signaling

Abstract Peritoneal spread is the primary mechanism of metastasis of ovarian cancer, and survival of ovarian cancer cells in the peritoneal cavity as nonadherent spheroids and their adherence to the mesothelium of distant organs lead to cancer progression, metastasis, and mortality. However, the mechanisms that govern this metastatic process in ovarian cancer cells remain poorly understood. In this study, we cultured ovarian cancer cell lines in adherent and nonadherent conditions in vitro and analyzed changes in mRNA and protein levels to identify mechanisms of tumor cell survival and proliferation in adherent and nonadherent cells. EGFR or ERBB2 upregulated ZEB1 in nonadherent cells, which caused resistance to cell death and increased tumor-initiating capacity. Conversely, Forkhead box M1 (FOXM1) was required for the induction of integrin β1, integrin-α V, and integrin-α 5 for adhesion of cancer cells. FOXM1 also upregulated ZEB1, which could act as a feedback inhibitor of FOXM1, and caused the transition of adherent cells to nonadherent cells. Strikingly, the combinatorial treatment with lapatinib [dual kinase inhibitor of EGFR (ERBB1) and ERBB2] and thiostrepton (FOXM1 inhibitor) reduced growth and peritoneal spread of ovarian cancer cells more effectively than either single-agent treatment in vivo. In conclusion, these results demonstrate that FOXM1 and EGFR/ERBB2 pathways are key points of vulnerability for therapy to disrupt peritoneal spread and adhesion of ovarian cancer cells. Significance: This study describes the mechanism exhibited by ovarian cancer cells required for adherent cell transition to nonadherent form during peritoneal spread and metastasis.

Combining PARP with ATR inhibition overcomes PARP inhibitor and platinum resistance in ovarian cancer models

AbstractOvarian cancer (OVCA) inevitably acquires resistance to platinum chemotherapy and PARP inhibitors (PARPi). We show that acquisition of PARPi-resistance is accompanied by increased ATR-CHK1 activity and sensitivity to ATR inhibition (ATRi). However, PARPi-resistant cells are remarkably more sensitive to ATRi when combined with PARPi (PARPi-ATRi). Sensitivity to PARPi-ATRi in diverse PARPi and platinum-resistant models, including BRCA1/2 reversion and CCNE1-amplified models, correlate with synergistic increases in replication fork stalling, double-strand breaks, and apoptosis. Surprisingly, BRCA reversion mutations and an ability to form RAD51 foci are frequently not observed in models of acquired PARPi-resistance, suggesting the existence of alternative resistance mechanisms. However, regardless of the mechanisms of resistance, complete and durable therapeutic responses to PARPi-ATRi that significantly increase survival are observed in clinically relevant platinum and acquired PARPi-resistant patient-derived xenografts (PDXs) models. These findings indicate that PARPi-ATRi is a highly promising strategy for OVCAs that acquire resistance to PARPi and platinum.

BRCA1Promoter Methylation and Clinical Outcomes in Ovarian Cancer: An Individual Patient Data Meta-Analysis

AbstractBackgroundBRCA1 methylation has been associated with homologous recombination deficiency, a biomarker of platinum sensitivity. Studies evaluating BRCA1-methylated tubal and ovarian cancer (OC) do not consistently support improved survival following platinum chemotherapy. We examine the characteristics of BRCA1-methylated OC in a meta-analysis of individual participant data.MethodsData of 2636 participants across 15 studies were analyzed. BRCA1-methylated tumors were defined according to their original study. Associations between BRCA1 methylation and clinicopathological characteristics were evaluated. The effects of methylation on overall survival (OS) and progression-free survival (PFS) were examined using mixed-effects models. All statistical tests were 2-sided.Results430 (16.3%) tumors were BRCA1-methylated. BRCA1 methylation was associated with younger age and advanced-stage, high-grade serous OC. There were no survival differences between BRCA1-methylated and non–BRCA1-methylated OC (median PFS = 20.0 vs 18.5 months, hazard ratio [HR] = 1.01, 95% CI = 0.87 to 1.16; P = .98; median OS = 46.6 vs 48.0 months, HR = 1.02, 95% CI = 0.87 to 1.18; P = .96). Where BRCA1/2 mutations were evaluated (n = 1248), BRCA1 methylation displayed no survival advantage over BRCA1/2-intact (BRCA1/2 wild-type non–BRCA1-methylated) OC. Studies used different methods to define BRCA1 methylation. Where BRCA1 methylation was determined using methylation-specific polymerase chain reaction and gel electrophoresis (n = 834), it was associated with improved survival (PFS: HR = 0.80, 95% CI = 0.66 to 0.97; P = .02; OS: HR = 0.80, 95% CI = 0.63 to 1.00; P = .05) on mixed-effects modeling.ConclusionBRCA1-methylated OC displays similar clinicopathological features to BRCA1-mutated OC but is not associated with survival. Heterogeneity within BRCA1 methylation assays influences associations. Refining these assays may better identify cases with silenced BRCA1 function and improved patient outcomes.

p85β regulates autophagic degradation of AXL to activate oncogenic signaling

AbstractPIK3R2 encodes the p85β regulatory subunit of phosphatidylinositol 3-kinase and is frequently amplified in cancers. The signaling mechanism and therapeutic implication of p85β are poorly understood. Here we report that p85β upregulates the protein level of the receptor tyrosine kinase AXL to induce oncogenic signaling in ovarian cancer. p85β activates p110 activity and AKT-independent PDK1/SGK3 signaling to promote tumorigenic phenotypes, which are all abolished upon inhibition of AXL. At the molecular level, p85β alters the phosphorylation of TRIM2 (an E3 ligase) and optineurin (an autophagy receptor), which mediate the selective regulation of AXL by p85β, thereby disrupting the autophagic degradation of the AXL protein. Therapeutically, p85β expression renders ovarian cancer cells vulnerable to inhibitors of AXL, p110, or PDK1. Conversely, p85β-depleted cells are less sensitive to these inhibitors. Together, our findings provide a rationale for pharmacological blockade of the AXL signaling axis in PIK3R2-amplified ovarian cancer.

Phase Ib Dose Expansion and Translational Analyses of Olaparib in Combination with Capivasertib in Recurrent Endometrial, Triple-Negative Breast, and Ovarian Cancer

Abstract Purpose: On the basis of strong preclinical rationale, we sought to confirm recommended phase II dose (RP2D) for olaparib, a PARP inhibitor, combined with the AKT inhibitor capivasertib and assess molecular markers of response and resistance. Patients and Methods: We performed a safety lead-in followed by expansion in endometrial, triple-negative breast, ovarian, fallopian tube, or peritoneal cancer. Olaparib 300 mg orally twice daily and capivasertib orally twice daily on a 4-day on 3-day off schedule was evaluated. Two dose levels (DL) of capivasertib were planned: 400 mg (DL1) and 320 mg (DL-1). Patients underwent biopsies at baseline and 28 days. Results: A total of 38 patients were enrolled. Seven (18%) had germline BRCA1/2 mutations. The first 2 patients on DL1 experienced dose-limiting toxicities (DLT) of diarrhea and vomiting. No DLTs were observed on DL-1 (n = 6); therefore, DL1 was reexplored (n = 6) with no DLTs, confirming DL1 as RP2D. Most common treatment-related grade 3/4 adverse events were anemia (23.7%) and leukopenia (10.5%). Of 32 evaluable subjects, 6 (19%) had partial response (PR); PR rate was 44.4% in endometrial cancer. Seven (22%) additional patients had stable disease greater than 4 months. Tumor analysis demonstrated strong correlations between response and immune activity, cell-cycle alterations, and DNA damage response. Therapy resistance was associated with receptor tyrosine kinase and RAS-MAPK pathway activity, metabolism, and epigenetics. Conclusions: The combination of olaparib and capivasertib is associated to no serious adverse events and demonstrates durable activity in ovarian, endometrial, and breast cancers, with promising responses in endometrial cancer. Importantly, tumor samples acquired pre- and on-therapy can help predict patient benefit.

Phase 1 trial of nelfinavir added to standard cisplatin chemotherapy with concurrent pelvic radiation for locally advanced cervical cancer

BackgroundNelfinavir (NFV), an HIV‐1 protease inhibitor, has been shown to sensitize cancer cells to chemoradiation (CRT). The objectives of this phase 1 trial were to evaluate safety and identify the recommended phase 2 dose of NFV added to concurrent CRT for locally advanced cervical cancer.MethodsTwo dose levels of NFV were evaluated: 875 mg orally twice daily (dose level 1 [DL1]) and 1250 mg twice daily (DL2). NFV was initiated 7 days before CRT and continued through CRT completion. Toxicity, radiographic responses, and pathologic responses were evaluated. Serial tumor biopsies (baseline, after NFV monotherapy, on NFV + CRT, and posttreatment) were evaluated by immunohistochemistry, NanoString, and reverse‐phase‐protein‐array analyses.ResultsNFV sensitized cervical cancer cells to radiation, increasing apoptosis and tumor suppression in vivo. Patients (n = 13) with International Federation of Gynecology and Obstetrics stage IIA through IVA squamous cell cervical carcinoma were enrolled, including 7 patients at DL1 and 6 patients at DL2. At DL1, expansion to 6 patients was required after a patient developed a dose‐limiting toxicity, whereas no dose‐limiting toxicities occurred at DL2. Therefore, DL2 was established as the recommended phase 2 dose. All patients at DL2 completed CRT, and 1 of 6 experienced grade 3 or 4 anemia, nausea, and diarrhea. One recurrence was noted at DL2, with disease outside the radiation field. Ten of 11 evaluable patients remained without evidence of disease at a median follow‐up of 50 months. NFV significantly decreased phosphorylated Akt levels in tumors. Cell cycle and cancer pathways also were reduced by NFV and CRT.ConclusionsNFV with CRT is well tolerated. The response rate is promising compared with historic controls in this patient population and warrants further investigation.

Multiomic analysis of cervical squamous cell carcinoma identifies cellular ecosystems with biological and clinical relevance

Cervical squamous cell carcinoma (CSCC) exhibits a limited response to immune-checkpoint blockade. Here we conducted a multiomic analysis encompassing single-cell RNA sequencing, spatial transcriptomics and spatial proteomics, combined with genetic and pharmacological perturbations to systematically develop a high-resolution and spatially resolved map of intratumoral expression heterogeneity in CSCC. Three tumor states (epithelial-cytokeratin, epithelial-immune (Epi-Imm) and epithelial senescence), recapitulating different stages of squamous differentiation, showed distinct tumor immune microenvironments. Bidirectional interactions between epithelial-cytokeratin malignant cells and immunosuppressive cancer-associated fibroblasts form an immune exclusionary microenvironment through transforming growth factor β pathway signaling mediated by FABP5. In Epi-Imm tumors, malignant cells interact with natural killer and T cells through interferon signaling. Preliminary analysis of samples from a cervical cancer clinical trial ( NCT04516616 ) demonstrated neoadjuvant chemotherapy induces a state transition to Epi-Imm, which correlates with pathological complete remission following treatment with immune-checkpoint blockade. These findings deepen the understanding of cellular state diversity in CSCC.

ZBP1 antagonizes MRE11-mediated DNA end resection and confers synthetic lethality to PARP inhibition in ovarian cancer

ZBP1, a classic pattern recognition receptor (PRR), has been implicated in regulating programmed cell death and the innate immune response. However, the role of ZBP1 in the nucleus remains largely undefined. Here, we found that nuclear ZBP1 localizes to the site of DNA double-stranded breaks (DSBs) following DNA damage and impairs homologous recombination (HR) repair through its interaction with MRE11. ZBP1 interacts with MRE11 through RHIM A and B domains and inhibits the enzymatic activity of MRE11, ultimately leading to the suppression of HR and DNA damage repair (DDR). These processes are initiated via ATM-mediated ZBP1 phosphorylation at S106. Consistent with these findings, in vitro and in vivo models both exhibit increased sensitivity to PARP inhibitor treatment following ZBP1 overexpression. Furthermore, in our neoadjuvant niraparib monotherapy study (NCT05407841) higher ZBP1 expression correlates with better response to PARP inhibition and prolonged PFS in high-grade serous ovarian cancer (HGSOC). This study describes a novel function of ZBP1 for regulating HR, which confers synthetic lethality to PARP inhibition in ovarian cancer. ZBP1 thus serves as a potential therapy target and biomarker of response to PARP inhibitors and potentially other therapeutic agents such as platin analogs that are synthetically lethal with defective HR.

Mitigating T cell DNA damage during PARP inhibitor treatment enhances antitumor efficacy

Poly(ADP-ribose) polymerase inhibitors (PARPis) are a class of agents targeting DNA damage repair that have become standard therapy for epithelial ovarian cancer (EOC) and multiple other solid tumors. In addition to targeting DNA damage repair, PARPis actively modulate antitumor immune responses, with efficacy being partially dependent on T cell activity. Here, we found that patient T cells sustain DNA damage during PARPi treatment, which reduces treatment efficacy. Leveraging paired pre- and posttreatment tumor samples from a clinical trial of patients with EOC treated with neoadjuvant niraparib as monotherapy, we showed that the PARPi caused DNA damage, slowed proliferation, and increased apoptosis in T cells, which we validated both in vitro and in mouse models. A genome-wide CRISPR (clustered regularly interspaced short palindromic repeats) knockout screen in primary human T cells identified PARP1 as the principal mediator of PARPi-induced T cell death. T cell–specific deletion of PARP1 or mutating Parp1 at its binding sites in transgenic mice led to reduced T cell DNA damage during PARPi treatment, resulting in improved efficacy of PARPis, alone or in combination with immune checkpoint inhibition. We then engineered PARPi-tolerant CAR T cells using cytosine base editing, which decreased PARPi-induced PARP1 trapping and led to reduced PARPi-induced DNA damage, resulting in superior antitumor efficacy in xenograft models compared with parental CAR T cells. This study highlights the relevance of PARPi-induced DNA damage to T cells and suggests opportunities to improve the efficacy of PARPis as monotherapy or in combination with immunotherapy.

Combination Therapy with Copanlisib and Niraparib in Patients with Recurrent Endometrial and Ovarian Cancer (COPANIRA): Efficacy, Toxicity, and Translational Insights

Abstract Purpose: Patients with recurrent endometrial or ovarian cancer have poor survival outcomes. We evaluated the clinical efficacy and toxicity of copanlisib [a phosphatidylinositol 3-kinase (PI3K) inhibitor] and niraparib [a poly (ADP-ribose) polymerase inhibitor (PARPi)] in this patient population with translational insights. Patients and Methods: This was a phase Ib trial. Copanlisib was administered intravenously on days 1, 8, and 15 of a 28-day cycle, and niraparib was given orally once daily. Four dose levels were explored over a dose-limiting toxicity (DLT) window of 28 days. The primary objective was to determine the recommended phase II dose (RP2D) of this combination. Secondary objectives included safety, objective response rate (ORR), and pharmacokinetics. Tumor biopsies were analyzed using reverse phase protein array (RPPA) to identify molecular correlates of response. Results: Thirty patients were enrolled. An RP2D was not established due to DLTs, most commonly a grade 3 maculopapular rash attributed to copanlisib. The ORR was 12.5% (95% confidence interval, 2.8%–33.6%). RPPA was performed on tumors from eight patients. PI3K pathway activity did not correlate with PI3K mutational status. Nineteen proteins were differentially expressed between patients with stable disease and those with progressive disease; many were substrates of Akt (protein kinase B), implicating downstream PI3K signaling in response. Conclusions: The combination of copanlisib and niraparib demonstrated limited tolerability, and the ORR was modest. However, functional proteomic analyses identified candidate biomarkers—particularly Akt pathway substrates—which may inform future strategies to optimize PI3K and PARPi combinations.

Clinical Studies of Endometrial Cytology and Cervical Methylation Assays in Endometrial Cancer Screening and Fertility-Preservation Evaluation

The current study aims to assess high-risk patients using both liquid-based cytology and cervical methylation testing. The results will be compared with the traditional hysteroscopic pathological findings to determine the sensitivity and specificity of these methods for early detection of endometrial cancer, thereby evaluating their potential application in early screening. Primary Objectives： 1. To evaluate the sensitivity, specificity, and accuracy of endometrial cytology for screening endometrial cancer. 2. To assess the sensitivity, specificity, and accuracy of methylation testing for screening endometrial cancer. 3. To perform further molecular testing on tissue samples obtained from endometrial cytology and cervical methylation tests, aiming to explore early screening-sensitive indicators. Secondary Objectives： 1. To determine the value of endometrial cytology in evaluating the efficacy of fertility-sparing treatments for endometrial cancer. 2. To assess the value of methylation testing in evaluating the efficacy of fertility-sparing treatments for endometrial cancer.

Pd-1 Antibody Combined Neoadjuvant Chemotherapy for Locally Advanced Cervical Cancer

Cervical cancer is one of the major health problems for chinese women. Besides surgery and radiotherapy, neoadjuvant chemotherapy has been proved to be an effective program by many studies. However, not all patients respond well to neoadjuvant chemotherapy. This is an open-label, single-arm, multi-center clinical trial to evaluate whether PD-1 in combination with neoadjuvant chemotherapy will achieve better objective response rate.