The nuclear pore complex (NPC) component TPR has emerged as a multifunctional scaffold implicated in mitosis, chromatin organization, mRNA export, and genome stability. However, TPR's role in mitogenic signal transduction remains largely unexplored. Here, we investigate whether nucleoporin TPR functions as a MAPK-regulated nuclear component that modulates mitogenic signals initiated at the plasma membrane-including EGFR activation-and their transcriptional output. Transcriptomic profiling reveals that TPR depletion reshapes EGF-induced, MAPK-responsive gene expression, including altered expression of MAPK pathway components and enhanced induction of the immediate-early gene FOS. Functionally, TPR-depleted cells exhibit increased FOS induction upon EGF stimulation and altered EGF-driven cell-cycle progression. Using a novel phospho-specific monoclonal antibody, we show that TPR is phosphorylated at Ser2155 following EGFR activation via the canonical RAS-RAF-MEK-ERK MAPK cascade, placing TPR downstream of MAPK pathway activation. This phosphorylation is suppressed by clinically used EGFR and BRAF inhibitors and, conversely, is constitutively induced by oncogenic RAS and BRAF, indicating that Ser2155 phosphorylation reflects MAPK pathway activity. In vivo, CRISPR/Cas9-engineered Tpr haploinsufficient mice show changes in MAPK pathway regulatory gene expression in bulk spleen RNA-seq, consistent with findings in human cells, and enhanced Fos induction in splenocytes upon CD3/CD28 stimulation, together suggesting a conserved association between TPR levels and altered MAPK-related transcriptomic profiles. Finally, immunohistochemical analysis reveals elevated TPR phosphorylation in serous ovarian carcinoma and heterogeneous phosphorylation patterns in triple-negative breast cancer, two tumor types frequently characterized by MAPK pathway hyperactivation. Together, these findings uncover a previously unappreciated role for TPR as a MAPK-responsive nuclear factor and support a model in which NPC-associated components fine-tune mitogen-induced transcriptional responses.

Detecting chromosomal copy-number alterations together with protein-defined cell states in intact tissue is critical for understanding early clonal evolution and microenvironmental interactions in cancer. We developed ORION-FISH, which integrates high-plex tissue imaging with a morphology-preserving DNA-FISH workflow and single-cell registration, yielding measurements concordant with clinical FISH. In High Grade Serous Ovarian Carcinoma (HGSOC), ORION-FISH recapitulated known chromosomal changes while revealing subclonal heterogeneity missed by targeted sequencing. Applied to serous tubal intraepithelial carcinomas (STICs), precursors of HGSOC, ORION-FISH identified intermixed epithelial cells with MYC or CCNE1 copy-number gains, as well as concurrent alterations associated with distinct immune microenvironments. In addition, epithelial cells with MYC and CCNE1 copy-number gains were detected in morphologically normal fallopian tube epithelium, along with rare MDM4 increases across epithelial lineages. Together, ORION-FISH provides a framework linking chromosomal copy number states to protein-defined phenotypes within preserved tissue architecture, enabling context-aware interrogation of early copy-number diversification at single-cell resolution.

Abstract Background: Platinum-resistant ovarian cancer (PROC) and clear cell gynecologic cancer (CCGC) are associated with poor prognoses and have limited effective treatment options. PROC has a recurrence rate of approximately 70%, while CCGC, a rare histologic subtype, demonstrates resistance to most standard therapies. These unmet clinical needs underscore the importance of developing novel therapeutics to improve patient outcomes. Immune checkpoint inhibitors have shown promising preliminary antitumor activity in PROC and CCGC, although they are not currently approved for either indication. Lorigerlimab is an investigational bispecific (PD-1 x CTLA-4), tetravalent DART® molecule. It is engineered to enhance CTLA-4 blockade in a PD-1-binding-dependent manner, intended to improve activity in the tumor microenvironment while minimizing off-tumor toxicity. Preclinical and early-phase clinical data support lorigerlimab immunomodulatory activity and tolerability. This study investigates the efficacy and safety of lorigerlimab monotherapy in patients with PROC and CCGC. Methods: This open-label, multicohort, multi-institutional, phase 2 study (NCT06730347) evaluates lorigerlimab monotherapy in participants with PROC (cohort A) or CCGC (cohort B). Cohort A utilizes a Simon’s two-stage design to enroll up to 40 participants and cohort B will enroll up to 20 participants. Participants receive lorigerlimab intravenously every 3 weeks for up to 35 cycles or until disease progression, unacceptable toxicity, or withdrawal. The primary objective is to evaluate the antitumor activity as measured by objective tumor response. Secondary objectives include characterization of safety, tolerability, and evaluation of efficacy by duration of response, disease control rate, progression-free survival, and change in tumor size from baseline. Eligible participants must have persistent or recurrent high-grade serous ovarian carcinoma or clear cell gynecologic cancers with at least 50% clear cell histomorphology. Participants with PROC must have received at least one and up to three prior lines of systemic therapy, which may include prior bevacizumab. Individuals with a germline or somatic BRCA mutation must have received PARP inhibitor therapy. Participants with CCGC must have received at least one prior line. All participants must have good functional status, measurable disease, adequate organ function, and no contraindications to immunotherapy. Individuals with primary platinum-refractory disease are excluded. Prior use of checkpoint inhibitor therapy is permitted for participants with clear cell endometrial or clear cell cervical cancer. Tumor assessments occur every 9 weeks for the first 54 weeks, then every 12 weeks until treatment discontinuation. Enrollment commenced in May 2025 and is ongoing. Citation Format: Helen D. Clark, Kelly M. Rangel, Shawana N. Richard, Jichao Sun, Pepi Pencheva, Chad Hamilton, Amir A. Jazaeri. A phase 2 multicohort study to evaluate lorigerlimab (PD-1 x CTLA-4) in participants with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(8_Suppl):Abstract nr CT279.

Natural killer (NK) cells contribute to tumor immunosurveillance, yet their heterogeneity across cancer types remains incompletely understood. Transcriptomic, spatial, and functional assays reveal that non-small cell lung carcinoma (NSCLC) is enriched in NK cells that mediate clinically relevant effector functions, whereas high-grade serous ovarian carcinoma (HGSOC) contains dysfunctional NK cells that express co-inhibitory receptors including NKG2A. Analysis of HGSOC patient samples and syngeneic mouse models indicates a crosstalk between NK cells and CD8⁺ T cells critical for effective antitumor immunity. Depletion of either population leads to phenotypic impairment of the reciprocal one. Blocking NKG2A restores NK cell cytotoxicity and promotes CD8⁺ T cell responses, significantly improving the efficacy of PD-1 blockade in murine HGSOC models. Thus, NK cells and CD8⁺ T cells engage in a functional interplay of immunological relevance. Moreover, the NKG2A-HLA-E axis represents a clinically actionable immunological checkpoint in tumors with impaired NK cell functions.

Background: High-grade serous ovarian carcinoma (HGSOC) is the most common and lethal subtype of epithelial ovarian cancer and is frequently diagnosed at advanced stages. Because currently available blood-based biomarkers have limited performance in early-stage disease, there is a need to identify circulating biomarker candidates associated with early-stage HGSOC. In this retrospective multi-institutional case-control study, we evaluated whether serum extracellular vesicle (EV)-associated protein signatures distinguish early-stage HGSOC from healthy controls. Methods: Serum samples (n = 252) were obtained retrospectively from multiple institutions and included healthy controls and patients with early- and advanced-stage HGSOC. EV-associated proteins were profiled using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and proximity extension assay (PEA) to identify candidate proteins enriched in early-stage HGSOC. Selected candidates were evaluated by enzyme-linked immunosorbent assay (ELISA), and tissue-level expression was examined in early-stage HGSOC specimens. A multimarker combination model was generated using a smoothed empirical estimate of hyper-volume under the manifold (SHUM) approach and internally assessed by leave-one-out cross-validation. Results: Ten EV-associated serum proteins were prioritized on the basis of differential expression and fold change and were confirmed to be expressed in early-stage HGSOC tissues. In ELISA-based analyses, the combined 10-protein EV panel distinguished early-stage HGSOC from healthy controls with an area under the curve (AUC) of 0.99 in the study dataset, whereas MUC16 (CA-125) showed substantially lower performance in this comparison. The SHUM-based model yielded a true-positive rate of 0.971, a false-positive rate of 0.057, and a Matthews correlation coefficient of 0.915 in the analyzed cohort. Several candidate proteins were differentially enriched in EV fractions but not in matched whole serum. Conclusions: Serum EV-associated proteins are altered in early-stage HGSOC and define a multi-protein signature associated with this disease state in a retrospective case-control setting. These findings support further evaluation of EV-based biomarker candidates in clinically representative and prospectively collected cohorts that include benign gynecologic conditions, symptomatic patients, and pre-diagnostic samples.

High-grade serous ovarian carcinoma (HGSC) is the sixth leading cause of cancer-related death among women. Most tumors arise from the Fallopian tubal epithelium (TE), exhibit numerous mutations, and present heterogenous pathological features. However, the contribution of specific mutation combinations to cellular transformation, pathological phenotype and chemotherapeutic responses remains unclear. We used a mouse TE organoid platform for combinatorial CRISPR mutagenesis of 20 candidate HGSC driver genes. Besides Trp53, mutations in Nf1, Cdkn2a and Map2k4 were the most prevalent in phenotypically transformed organoids. Map2k4 mutant organoids transplanted into mice predominantly yielded papillary-glandular carcinomas, whereas those with Nf1 mutations were more mesenchymal-like. Map2k4 mutant cells were particularly sensitive to paclitaxel, and Rho kinase inhibitor (ROCKi) increased trametinib sensitivity in both Map2k4- and Nf1-mutant organoids. This organoid mutagenesis strategy is powerful for unraveling the genetic and phenotypic complexity of HGSC, and identified Map2k4 as a potential therapeutic target in select HGSC cases.

Objective: To determine the frequency and clinicopathological features of incidental premalignant and malignant gynecological lesions detected after hysterectomies performed for benign indications, and to identify associated risk factors. Methodology: This retrospective study reviewed 1,047 hysterectomies performed for benign conditions at a tertiary center in Ankara, Turkiye, over a ten years period (January 2006-December 2015). Demographic characteristics, preoperative assessments, surgical indications, and histopathological outcomes were analyzed. Incidental lesions were defined as pre-malignant or malignant pathologies identified postoperatively without prior clinical suspicion. Statistical analyses were performed using chi-square and t-tests. Results: Incidental pre-malignant or malignant lesions were identified in 6% (n=63) of cases, including cervical dysplasia/HSIL (0.9%), borderline ovarian tumors (1.1%), endometrial adenocarcinoma (0.5%), leiomyosarcoma (0.5%), and high-grade serous ovarian carcinoma (0.6%). Postmenopausal women had a significantly higher incidence than premenopausal women (9.2% vs. 3.8%, p<0.001). Patients with incidental findings were older than those with benign pathology (55.4 ± 11.2 vs. 52.7 ± 9.6 years, p=0.034). Larger myomas were associated with uterine sarcoma (172 ± 92 mm vs. 71 ± 39 mm, p<0.001). Ovarian malignancies were detected in 2.4% of cases without suspicious ultrasound features and in 14.8% of cases with ≥2 malignancy criteria (p=0.013). Serous tubal intraepithelial carcinoma (STIC) was identified in 0.28% (n=3). Conclusion: Incidental pre-malignant or malignant lesions were present in 6% of hysterectomies performed for benign indications. Age, menopausal status, myoma size, and suspicious imaging features were significant predictors. Preoperative evaluation may not completely exclude the possibility of occult pathology; therefore, this risk should be discussed during preoperative counseling.

This study reports a rare case of mixed ovarian carcinoma composed of squamous cell carcinoma (SCC) and high-grade serous carcinoma (HGSC) arising from endometriosis, and provides a systematic review of the relevant literature. A 59-year-old female presented with bilateral ovarian cystic lesions (4.6cm on the left, 9.6cm on the right) and a mural nodule in the right ovarian cyst. Serum tumor markers were CA125 57.50 U/mL and CA19-9 6.74 U/mL. The patient underwent total hysterectomy, bilateral salpingo-oophorectomy, omentectomy, and appendectomy. Histopathological examination revealed a mixed carcinoma of the right ovary composed of SCC and HGSC, with adjacent serous borderline tumor and endometriotic cyst. The left ovary showed a serous borderline tumor with endometriosis. Next-generation sequencing (NGS) identified two distinct TP53 mutations: a missense mutation in the HGSC component and a splice-site mutation in the SCC component. The patient received six cycles of paclitaxel and carboplatin chemotherapy. Within two months after completing the treatment, tumor markers had normalized and no recurrence was detected. We describe a 59-year-old woman diagnosed with stage IA mixed ovarian carcinoma (SCC and HGSC), in which two distinct TP53 gene mutations were identified. A systematic literature review was conducted using PubMed, Embase, and Web of Science databases. A total of eight published cases of ovarian mixed carcinoma containing a squamous component were identified. Of these, five originated from endometriosis and one from a mature cystic teratoma. Histologically, five cases were endometrioid adenocarcinoma with squamous differentiation, while others included clear cell carcinoma, mucoepidermoid carcinoma, and HGSC (the present case), each admixed with SCC components. This is the first reported case of ovarian HGSC coexisting with SCC. Given its extreme rarity, standardized treatment strategies have not yet been established.

Folate receptor 1 (FOLR1) has recently become a well-accepted therapeutic target in advanced-stage cancers. In this study, the prevalence of FOLR1 expression across all types of gynecologic tumors was investigated and correlated with selected clinicopathologic features. A total of 306 gynecologic tumors from 304 patients were evaluated for FOLR1 expression by immunohistochemistry (IHC). A positive FOLR1 is defined as ≥75% of viable tumor cells with moderate to strong membrane staining. Of 306 tumors, 31 (10.1%) had positive FOLR1 tests; a large majority of these FOLR1-positive tumors were HGSCs (64.5%), followed by uterine serous carcinoma, poorly differentiated/high-grade carcinoma, ovarian endometrioid carcinoma, ovarian mixed carcinoma, ovarian low-grade serous carcinoma, and serous borderline tumor with cribriform and micropapillary features. FOLR1 overexpression correlated with positive PD-L1 expression (P=0.012), intact mismatch repair protein (MMR) expression (P=0.024), and positive ER expression (P=0.040). In endometrial tumors, positive FOLR1 expression was associated with poor histologic grade (P=0.019), larger tumor size (P=0.048), mutant p53 expression (P<0.001), and lower PR expression (P=0.015). Endometrial tumors with FOLR1 overexpression had a significantly higher rate of TP53 mutations (P=0.013), while all endometrial tumors with PTEN alterations were negative for FOLR1 (P=0.037). Overall, FOLR1 overexpression was associated with poor prognostic factors, such as advanced clinical stage, increased recurrence rate, higher pathologic T and N stage, poor histologic grade, larger tumor size, lymphovascular invasion, uterine serosa involvement, and shorter progression-free survival.

Kinesin family member 15 (KIF15) is a molecular motor protein that participates in bipolar spindle assembly and centrosome separation during metaphase in normal cells. However, accumulating evidence has shown that KIF15 is aberrantly regulated in the state of malignancy and is associated with the progression of cancers. We analyzed the mRNA expression, protein expression, single-cell gene expression, prognoses, and immune reactivities of KIF15 using the data from various sources, including the Cancer Cell Line Encyclopedia, The Cancer Genome Atlas database, the Clinical Proteomic Tumor Analysis Consortium Confirmatory/Discovery database, and Cancer Single-cell Expression Map. Additionally, immunohistochemistry was performed to validate KIF15 protein expression in human tumor samples. The data from the present pan-cancer analysis elucidated that KIF15 expression was increased in more than 25 human tumors. Furthermore, it was found to be highly corre-lated with biological pathways, immune cell infiltration, and poor prognoses across multiple cancers. Additionally, tissue microarray analysis confirmed abundant cytoplasmic KIF15 expression in ovarian serous carcinoma and colorectal adenocarcinoma, with significantly higher levels compared to adjacent normal tissues. Consistent patterns of high KIF15 expression were also evident across various cancer cell lines in the CCLE dataset. Collectively, these observations suggest KIF15 is in-timately involved in the progression of multiple human cancers. By analyzing both the pro-teins that interact with KIF15 and the genes that show correlation with its expression, KEGG pathway enrichment indicated that processes such as cell cycle regulation, gap and tight junction dynamics, involvement of microRNAs in cancer, and the p53 signaling pathway may underlie the role of KIF15 in cancer development. KIF15 may serve as a potential biomarker and oncogenic factor in multiple cancer types.

Low-grade serous ovarian carcinoma is a rare subtype of epithelial ovarian cancer, accounting for less than 10% of serous malignancies. Compared to high-grade serous ovarian carcinoma, it follows an indolent course, often affects younger women, and demonstrates resistance to conventional cytotoxic chemotherapy. Low-grade serous ovarian is characterized by recurrent MAPK pathway alterations (KRAS, BRAF, NRAS) and frequent expression of functional hormone receptors, supporting the rationale for targeted and endocrine strategies. Optimal management relies on complete surgical cytoreduction. Endocrine therapy has shown promise, particularly in maintenance settings, and is being investigated in frontline strategies. MEK inhibitors, especially trametinib and avutometinib in combination with defactinib, have recently demonstrated improved outcomes in recurrent disease, while new combination strategies are under active evaluation to overcome resistance mechanisms. Immunotherapy remains of limited efficacy, though biomarker-driven combinations are explored. Ongoing biomarker-guided trials are expected to refine treatment paradigms.

PRSS23 promotes ovarian cancer peritoneal dissemination independent of protease activity.

Ovarian high-grade serous carcinoma (ovarian HGSC) is a clinically challenging disease with a poor prognosis, particularly for patients receiving neoadjuvant chemotherapy (NACT) before debulking surgery. In this study, we evaluate the progression-free interval (PFI) after NACT based on hematoxylin and eosin-stained whole-slide images (WSIs) of omental tumor tissue. Digital pathology tools are emerging, aiming at assisting pathologists in diagnosis and analysis; however, distinguishing features associated with response to NACT remain elusive. Multiple instance learning (MIL) coupled with attention mechanisms has shown promise in predicting treatment response from WSIs. Additionally, segmentation tools can identify and delineate regions in WSIs. Whereas some efforts have been made to develop explainable models for clinical outcome, there remains a need for genuinely interpretable models for pathologists. This article introduces the PATHOS framework, a novel approach to explaining crucial features of treatment response based on the PFI time in NACT treated patients from WSIs. PATHOS is composed of three blocks: (1) MIL block to identify informative regions, (2) panoptic segmentation and downstream analysis block for feature computation, and (3) classification block to predict the PFI. The results demonstrate that PATHOS enhances the interpretability of response to NACT in ovarian HGSC patients by highlighting pathologically significant features relevant to PFI prediction, such as tumor cell morphology, stromal abundance, and the spatial distribution of stromal regions. Furthermore, PATHOS identifies approximately 10% of the total WSI area as an informative region for clinical outcome.

Ovulation-derived extracellular vesicles exhibit sustained oncogenic influence on the exposed fallopian tube fimbrial cells after drainage into peritoneal cavity.

Early detection of ovarian cancer requires the observation of subtle changes within the fallopian tubes, where serous tubal intraepithelial carcinoma lesions, the putative precursor of high-grade ovarian serous carcinoma, may be present and detectable. The cell-acquiring fallopian endoscope was designed to detect and interrogate potentially pathological sites in the fallopian tubes via alterations in fluorescence signal and the collection of epithelial cells. We performed a comprehensive redesign of a first-generation endoscope prototype and demonstrated its performance in whole, human ex vivo fallopian tubes. Through the iterative improvements to the design, the endoscope features improved imaging, cell collection, and general ease of use. Specifically, improvements included a larger core count fiber imaging bundle, a redesigned close-focus lens, more flexible materials, an altered cell collection method, and a lighter-weight handle. With benign fallopian tube samples from three patients, we demonstrate cell collection on the order of cells per collection and imaging capabilities that result in average image intensity ratios. This second-generation endoscope is suitable for the study of intact fallopian tubes.

BackgroundFor patients with advanced ovarian cancer undergoing cytoreductive surgery, achieving optimal surgical outcomes has significant clinical implications for the design of adjuvant therapy and surveillance strategies. This retrospective, single-center cohort study aimed to develop a radiomics model based on preoperative PET-CT imaging parameters to predict optimal cytoreductive surgery outcomes, thereby providing clinical decision-making support to improve the prognosis of patients with advanced ovarian cancer.Material/MethodsPatients with clinically staged IIIC-IVB high-grade serous ovarian carcinoma (HGSOC) who underwent primary cytoreductive surgery between October 2020 and October 2023 were enrolled and divided into training and validation cohorts. A radiomics model based on preoperative PET-CT was developed, and its performance was quantitatively evaluated and compared within the validation cohort. Subsequently, the PET-CT radiomics model was combined with the Suidan score to generate a combined model.ResultsOptimal cytoreduction rates were 72.86% (training) and 75.0% (validation). The PET-CT radiomics model demonstrated superior discriminative ability (AUC: 0.808, 95%CI: 0.665–0.951) compared to the Suidan score (AUC: 0.773, 95%CI: 0.615–0.931) in validation. The combined model achieved the highest predictive performance (AUC: 0.836, 95%CI: 0.706–0.966), with sensitivity 81.8%, specificity 83.9%, PPV 64.3%, NPV 92.9%, and accuracy 83.3%.ConclusionsThe combined model integrating PET-CT radiomics and Suidan score provides accurate preoperative prediction of cytoreductive outcomes, optimizing treatment strategies for advanced ovarian cancer.

Introduction: Ovarian cancer is one of the most common gynecological cancers in the United States. Common sites of distant metastasis from ovarian cancers and other cancers of Mullerian origin include the liver, pleura and lungs. However, metastasis to the brain remains exceptionally rare, ranging from 0.49 to 6.1%. Hence, the scarcity of such cases poses significant diagnostic and management challenges. Case Presentation: We present a case of an 80-year-old female who at the time of initial diagnosis presented with complaints of right leg pain, shortness of breath and cough. Imaging studies were remarkable for a pulmonary embolism, 2.5 cm mediastinal mass, pleural effusions, omental caking and an occlusive thrombus in the right greater saphenous vein. Malignancy was suspected in the setting of hypercoagulability. Biopsy of the omentum and pleural cytology revealed a high grade ovarian serous carcinoma. The patient received neoadjuvant chemotherapy followed by cytoreductive surgery and additional chemotherapy afterwards. She demonstrated good response to treatment with follow up PET without evidence of disease. Over the next four years, the patient was intermittently placed on chemotherapy when found to have elevated CA125 levels and PET scan showing small volume disease mostly in the pelvis. Six-years later, the patient presented to the oncology clinic with complaints of dizziness and imbalance for the past month. MRI brain showed a new left cerebellar mass with vasogenic edema and obstructive hydrocephalus. However, restaging CT chest, abdomen and pelvis showed minimal to no disease, with the only possible foci being a 1.2 cm paraaortic lymph node. The patient underwent left suboccipital craniotomy and cerebellar tumor resection with pathology showing metastatic carcinoma consistent with spread from a Mullerian primary. Conclusion: This case emphasizes the diagnostic complexity and evolving clinical course of Müllerian tumors. In a patient with a history of Mullerian tumor and new onset neurological symptoms, differential diagnosis should include metastasis to the brain, even with minimal to no active pelvic and systemic disease burden.

Drug resistance jeopardizes the prognosis of high-grade serous ovarian carcinoma (HGSOC) patients via DNA damage repair-coupled mechanism. The role of biomolecular phase separation in DNA damage repair has loomed. Here we find that CBX2 condensates are associated with drug resistance and contribute to DNA double-strand break (DSB) repair in HGSOC. Specifically, CBX2 condensates facilitate the recruitment of key DSB repair factors PARP1, 53BP1, and BRCA1 to chromatin. Patients with a CBX2-negative pattern exhibit the best prognosis, followed by those with non-condensate CBX2, while the worst outcomes are observed in patients with condensate CBX2. By drug screening, Ibrutinib is identified as an effective inhibitor of HGSOC cells and patient-derived organoids with CBX2 condensates. Overall, CBX2 phase separation enhances DSB repair-mediated drug resistance in HGSOC cells, and Ibrutinib may offer a viable therapeutic option for CBX2-positive HGSOC patients.

The development of folic acid-based radioconjugates has been proposed for imaging and targeted radionuclide therapy of folate receptor-α (FRα)-positive cancer. Although folic acid binds both the tumor-associated FRα and the folate receptor-β (FRβ) expressed on certain blood cells, 6S-5-methyltetrahydrofolate (6S-5-MTHF) was reported to exhibit higher affinity for FRα. The aim of this study was, therefore, to investigate whether previously developed 6S-5-MTHF-based radioconjugates would preferentially bind to FRα. Methods: The 6R- and 6S-5-MTHF-based folate radioconjugates [177Lu]Lu-RedFol-1, [177Lu]Lu-RedFol-3, [177Lu]Lu-RedFol-24, and [177Lu]Lu-RedFol-25 were evaluated invitro using transfected Chinese hamster ovary cells to assess their cell uptake and binding affinity to FRα expressed on RT16 cells and to FRβ expressed on D4 cells. Biodistribution and SPECT/CT imaging studies of the 5-MTHF-based radioconjugates were performed in a mouse model bearing both RT16 and D4 xenografts. Immunohistochemical staining was performed on selected patient-derived tissue sections of high-grade serous ovarian carcinoma, kidney, and bone marrow to provide a representative overview of FRα and FRβ expression. Results: The uptake and receptor binding affinity of 6R-5-MTHF-based radioconjugates were in the same range for RT16 and D4 cells, indicating comparable binding to the FRα and FRβ. The 6S-5-MTHF-based radioconjugates showed a 4-6-fold higher uptake in RT16 cells than in D4 cells, which can be ascribed to the 9-29-fold increased binding affinity to FRα than to FRβ. In vivo, the uptake of 6R-5-MTHF-based radioconjugates in RT16 and D4 xenografts reached 55%-73% IA/g and 25%-44% IA/g at 24 h after injection, respectively. The accumulation of 6S-5-MTHF-based radioconjugates in RT16 xenografts was 74%-100% IA/g, which was 9-21-fold higher than the 4%-10% IA/g uptake observed in D4 xenografts. Immunohistochemistry data confirmed high FRα expression in human ovarian cancer tissue sample and the presence of FRα in renal proximal tubules. In contrast, FRα was absent in human bone marrow, where FRβ was substantially expressed. Conclusion: This study confirmed that the use of 6S-5-MTHF-based radioconjugates can enable selective targeting of FRα. The high expression levels of FRα in ovarian cancer but absence in the bone marrow of human tissue samples infer therapeutic and safety benefits of using FRα-selective folate radioconjugates. Measures to reduce renal retention of 6S-5-MTHF radioconjugates may, however, still be necessary.

Recurrent high-grade serous ovarian carcinoma (HGSOC) and endometrial cancer (EC) remain major clinical challenges with limited effective treatment options. DHX9, a DNA/RNA helicase essential for genomic stability, has not yet been explored as a therapeutic target in gynecologic cancers. Here, we show that a selective DHX9 inhibitor (DHX9i) suppresses proliferation in a subset of HGSOC and EC cell lines by inducing DNA damage, chromosomal instability, and mitotic failure. This effect was independent of microsatellite instability status and prior resistance to platinum or PARP inhibitors. Genomic analysis indicated that DHX9i resistance was unlikely to be driven by single-gene mutations but was instead associated with copy-number alterations in mitotic spindle and microtubule-regulating genes in both HGSOC and EC. Transcriptomic profiling further revealed consistent alteration of microtubule- and spindle-associated pathways in DHX9i-resistant models following DHX9i treatment. Mechanistically, DHX9i induced mitotic defects in DHX9i-sensitive models, while resistant lines maintained mitotic integrity. Given the convergence of resistance-associated features on microtubule-related pathways, we combined DHX9i with the microtubule-stabilizing agent paclitaxel to enhance mitotic stress. This combination triggered mitotic disruption and enhanced cytotoxicity in DHX9i-resistant cells. In vivo, the combination led to sustained tumor regression and prolonged survival in both DHX9i-sensitive and -resistant models without notable toxicity. Overall, our findings define genomic, transcriptomic, and phenotypic characteristics associated with differential response to DHX9i and support clinical evaluation of DHX9i-paclitaxel combination as a therapeutic strategy in recurrent gynecologic cancers.