Inherited BAP1 mutations cause melanoma and other cancers and can also lead to white hair patches or skin spots due to pigment cell loss.

Ovarian cancer stem cells (CSCs) can seed recurrent drug-resistant disease. Likewise, non-CSCs can acquire CSC phenotypic properties. How this process is orchestrated is of interest to inform how it might be prevented. We tested the hypothesis that ovarian CSC and/or drug-resistant tumor cells confer stem-like properties via extracellular vesicles (EVs). We focused our investigation on how EVs might mediate EZH2 signaling to promote a phenotypic change in drug-sensitive, non-CSCs. To accomplish this, we utilized paired PARP inhibitor-sensitive and - resistant ovarian cancer (OvCa) cell lines, EZH2 knockdown lines, and patient-derived organoids (PDOs) originating from recurrent high-grade serous OvCa. Small EVs isolated from drug-sensitive, CSC and/or drug-resistant enriched cultures, PARP inhibitor (olaparib) resistant lines, or drug-treated (olaparib or carboplatin) lines were cultured with treatment naïve or sensitive lines for defined time points. The impact of small EV exposure was determined by assessing cell number, metabolic activity, viability, sphere and colony-forming capacity, ALDH activity, DNA damage, and changes in associated signaling pathways. We found that EVs from CSC or drug-resistant enriched cell fractions communicate CSC-like phenotypes to the more sensitive tumor cells via EZH2 canonical and non-canonical signaling pathways, promoting stemness. We conclude that EV-mediated activation of EZH2 signaling represents a targetable mechanism contributing to stemness-associated drug resistance in OvCa. Graphical abstract:

Treatment of patients with platinum-resistant ovarian cancer is a major clinical challenge. We found that high expression of a meiotic protein, Synaptonemal Complex Protein 2 (SYCP2), is associated with platinum resistance and tyrosine kinase ABL1 inhibitor sensitivity in ovarian cancer. We demonstrate that tyrosine kinase ABL1 inhibitors inhibit cancer cell proliferation more efficiently in ovarian cancer cell lines with SYCP2 overexpression. Moreover, ABL1 inhibition effectively prevents tumor growth in vivo. Mechanistically, we identified a phosphorylation motif [RK]-x(2,3)-[DE]-x(2,3)-Y in SYCP2 and found that abolishing ABL1-mediated phosphorylation of SYCP2 at its tyrosine (Y) 739 within this motif renders ABL1 sensitivity of cancer cells. Importantly, ABL1 and SYCP2 colocalize at sites of R-loops after damage and promote transcription-coupled homologous recombination. Moreover, ABL1-mediated Y739 phosphorylation of SYCP2 promotes function of SYCP2 at sites of R-loops by facilitating RAD51 localization and repair, contributing to ovarian cancer cell survival. Overall, these findings highlight a novel therapeutic mechanism where ABL1 inhibitors induce cell death in platinum-resistant ovarian cancer by impairing transcription-coupled homologous recombination repair.

Solid tumors present a significant challenge to Chimeric Antigen Receptor (CAR) -T cell therapy, primarily due to limited T-cell infiltration and persistence in the tumor microenvironment. Cancers with predominantly peritoneal metastasis like ovarian cancer pose a substantial trafficking challenge to systemically administered CAR-T cell therapy. To identify chemokines that may guide CAR-T cells to tumor sites, we evaluated chemokine expression in primary and metastatic tumor samples from patients with ovarian cancer by immunohistochemistry. After identifying CCL2 as the most common chemokine expressed in both primary and metastatic disease, we validated CCL2 levels in serum samples from these patients. We found that CCL2 and CCL4 were commonly expressed in several ovarian cancer cell lines, a patient-derived tumor cell line and patient serum samples in vitro. We engineered armed Muc16/CA-125-directed CAR-T cells with cognate receptors for CCL2 and CCL4 and showed significant in vitro and in vivo tumor-directed trafficking. Finally, intravenously administered CCL2-armored CAR-T cells significantly prolonged survival in peritoneal tumor bearing mice. In conclusion, we identified CCL2 as a commonly expressed chemokine in patients with local and metastatic ovarian cancer and successfully demonstrate that armed CAR-T cells with enhanced homing to CCL2-expressing ovarian cancer are more efficacious due to improved peritoneal trafficking in vivo.

UVR is known to be the most important environmental carcinogen for cutaneous melanoma. Whereas genomic analyses of melanoma tumors implicate a high rate of UV damage, the experimental induction and recovery of bona fide UV-signature changes have not been directly observed. To replicate recurrent UV variants from The Cancer Genome Atlas_SKCM specimens, we UV irradiated cultured immortalized human melanocytes and subjected them to in vivo tumorigenesis assays. Exome sequencing of the xenografted tumors revealed an increase in UV-signature mutations within the tumors and identified 48 induced variants that overlap with The Cancer Genome Atlas skin cutaneous melanoma UV-hotspot mutations. A UV-induced mutation, ZNF831 p.R1393Q, was correlated with a decreased survival (hazard ratio = 5.44, 95% confidence interval = 1.92-15.47, P = .0015) and was preferentially observed in melanomas compared with that in all The Cancer Genome Atlas tumors (P = 4.42 x 10_7). In addition, ZNF831 mRNA expression loss was strongly associated with decreased patient survival (hazard ratio = 2.14, 95% confidence interval = 1.62-2.83, P = 7.91 x 10_8), although the transcripts may arise from multiple cell types, including T cells. In multiple melanoma lines, overexpression of wild-type ZNF831 reduced spheroid growth, heightened apoptosis, and increased cell motility, with the ZNF831 p.R1393Q variant partially or wholly abolishing these functional phenotypes. We thus experimentally recovered a "functional UV-hotspot mutation" in ZNF831 that is altered in human melanoma specimens.

Bispecific T-cell Engagers (BITEs) are a novel form of immunotherapy that overcome a deficiency of immune checkpoint inhibitors (ICI) by targeting a preidentified tumor associated antigen and redirecting a polyclonal population of effector T-cells against the tumor. High grade serous ovarian cancer is a lethal disease in the recurrent setting and has not been amenable to ICI therapy. MUC16/CA125 is overexpressed in high grade serous ovarian cancer. BITEs targeting the tumor-retained portion of MUC16/CA125 have recently been described and are in early-phase clinical trials. To identify mechanisms of resistance to BITEs, we collected serum, peripheral blood mononuclear cells, and ascites samples from patients with disease progression on MUC16-directed bispecific antibodies. Analysis of these samples showed downregulation of MUC16/CA125, elevated secretion of VEGF, and epithelial-to-mesenchymal transition in tumor cells. Interestingly, hypoxia was determined to be a driver of these changes. These findings were prospectively validated in ovarian cancer cell lines with CRISPR/Cas9 knockout of MUC16/CA125 and VEGF. Peripheral blood mononuclear cells from patients with disease progression were capable of effective cytolysis ex vivo, suggesting that resistance to therapy was primarily tumor driven. Restoration of MUC16/CA125 expression did not restore cytotoxicity in the presence of increased VEGF secretion. Combination treatment with a VEGF inhibitor rescued cytotoxicity in hypoxia-conditioned ovarian cancer cell lines with preserved target antigen expression. Collectively, these data outline a link between hypoxia and the development of resistance to BITEs and posits inhibition of VEGF inhibition as a potentially important therapeutic intervention.

There have been several important advancements in the management of ovarian cancer. Antibody drug conjugates have been transformative for this disease, but knowledge on the mechanisms of resistance to this modality and potential therapeutic salvage options are limited. Emerging data suggest that antigen loss or downregulation may not be a major contributor to resistance to antibody drug conjugates. Bispecific T-cell Engagers have the potential to exploit the persistent antigen expression in antibody drug conjugate-resistant ovarian cancer by binding these targets and redirecting polyclonal T-cells to facilitate cytotoxicity. Bispecific T-cell engagers have been approved for the treatment of uveal melanoma, but testing in other solid tumors, such as ovarian cancer, is ongoing. We identified the tumor-retained portion of the cancer antigen Muc16/CA125, MUC16-ectodomian (Muc16ecto), as a suitable target for bispecific T-cell engager therapy. We developed antibodies against Muc16ecto and showed that bispecific T-cell engagers lead to cytotoxicity against ovarian cancer cells in vitro and in vivo. Even in the presence of shed CA125, bispecific engagers against Muc16ecto preferentially bound to the intended cell-retained target. Furthermore, we found that the efficacy of Muc16ecto bispecific engagers was increased when combined with either anti-PD1 antibodies or anti-VEGF antibodies, the latter providing greater efficacy. Bispecific engagers for ovarian cancer, including one targeting Muc16ecto, are undergoing evaluation in clinical trials. We collected samples from patients experiencing progression of disease on these bispecific engagers to understand mechanisms of resistance. We found Muc16ecto downregulation in tumor samples obtained from patients experiencing progression on Muc16ecto-directed bispecific engagers. Our data showed that the relatively hypoxic peritoneal tumor microenvironment drove this process. Peripheral T-cells from patients experiencing progression were still capable of cytotoxicity against cancer cells ex vivo in the presence of tumors with retained Muc16ecto expression. Serum from these patients suppressed cytotoxicity, suggesting that other factors besides antigen downregulation were involved. We found that downregulation of Muc16ecto led to an epithelial to mesenchymal transition of ovarian cancer cells and increased VEGF secretion. Soluble VEGF alone was enough to blunt Muc16ecto bispecific T-cell activity, and this was found to be due to decreased T-cell proliferation and increased T-cell apoptotic priming. Our data posit that tumor heterogeneity due to VEGF secretion from hypoxic progressing sites, leading to disease progression at other non-hypoxic metastatic sites, could be a significant contributor to therapeutic failure. Oladapo Yeku, Mengyao Xu, Syem K. Barakzai, Raj Kumar, Irva Veillard. Mechanisms and consequences of bispecific T-cell engager immunotherapy for ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr IA012.

Adoptive cell therapies (ACTs), such as chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEDs), offer promising immunotherapeutic strategies for ovarian cancer (OC) due to their robust cytolytic capacity and ability to establish long-term immune surveillance. However, despite encouraging preclinical data, clinical responses remain limited. This discrepancy highlights an urgent need to uncover tumor-intrinsic resistance mechanisms that may impair ACT efficacy in OC. To investigate mechanisms of immunotherapy (IT) resistance in OC, we developed OVCAR3-derived cell models resistant to Ipilimumab/Nivolumab in vivo (OV3 IE ) and to BiTEDs in vitro (OV3 R ), and established patient-derived organoids (PDOs) from IT responders and non-responders. Candidate mediators of resistance were identified via RNA sequencing of OV3 IE cells and validated by Western blot. Tumor cell sensitivity to granzyme/perforin (Gr/PFN), Muc16/CA-125 (4h11-28ζ) - targeting CAR T cells, and Muc16/CA-125 (4H11) - targeting BiTEDs was assessed in cytotoxicity assays using Incucyte live-cell imaging. The functional role of the extracellular matrix protein Nidogen-2 (NID2) in resistance was evaluated through gene overexpression, knockdown, and knockout. Western blot analyses of Ras and Bcl-xl signalling pathways were performed, and pharmacologic inhibitors targeting these pathways were used to test their potential to reverse immune resistance. IT-resistant cell lines and PDOs exhibited elevated NID2 expression. NID2 overexpression conferred resistance to Gr/PFN, 4h11-28ζ CAR T cells, and 4H11 BiTEDs, whereas knockdown or knockout restored tumor cell susceptibility. Mechanistically, NID2 upregulation enhanced Ras activation and Bcl-xl expression. Pharmacologic inhibition of either pathway - using the pan-Ras inhibitor ADT-007 or Bcl-xl inhibitors (Navitoclax, Venetoclax, A-1331852) - restored cytotoxicity in NID2-high models, including resistant PDOs. NID2 is a tumor-intrinsic driver of IT resistance in OC, acting through Ras activation and Bcl-xl upregulation. It may serve as a predictive biomarker for ACT response. Targeting this pathway, particularly via Bcl-xl inhibitors offers a mechanistically grounded and clinically actionable strategy to overcome immune resistance. Fabian BT. Kraus, Mengyao Xu, Raj Kumar, Irva Veillard, Will B. Manning, Timothy S. Bond, Eugene Kim, Yusuke Matoba, Maryam Azimi, Bastian Czogalla, Sven Mahner, Fabian Trillsch, Bo R. Rueda, David R. Spriggs, Oladapo O. Yeku. Anchored in Resistance: Overcoming Nidogen-2 (NID2) - mediated Immunoresistance in Ovarian Cancer with Ras and Bcl-xl Inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B086.

Melanoma is a highly metastatic form of skin cancer for which current therapies offer limited benefits. We show here that the histone reader ATAD2 is overexpressed in melanoma and predicts poor prognosis, and that the MAP kinase pathway, via the transcription factor E2F1, stimulates ATAD2 expression. Genetic or pharmacological inhibition of ATAD2 suppresses the growth and metastasis of BRAF and NRAS mutant melanoma. Mechanistically, we show that ATAD2 inhibition activates both distinct and common tumor-suppressive pathways in BRAF and NRAS mutant melanoma. In particular, we find that ATAD2 inhibition induces ferroptosis in both contexts by downregulating the ferroptosis suppressor GPX4. The ferroptosis inducer erastin also inhibits melanoma growth. Combining the ATAD2 inhibitor BAY-850 with the MEK inhibitor trametinib potently suppresses melanoma growth. Our study identifies ATAD2 as a key driver of melanoma and provides a rationale for targeting ATAD2 in conjunction with the MAPK pathway to treat melanoma.

Large-scale cancer genetic/genomic studies demonstrated that papillary renal cell carcinoma (pRCC) is featured with a frequent shallow deletion of the upstream tumor suppressors of the Hippo/YAP signaling pathway, suggesting that this signaling pathway may play a role in pRCC development. Here we develop a transgenic mouse model with a renal epithelial cell-specific hyperactivation of YAP1 and find that hyperactivation of YAP1 can induce dedifferentiation and transformation of renal tubular epithelial cells leading to the development of pRCC. We analyze at the single-cell resolution the cellular landscape alterations during cancer initiation and progression. Our data indicate that the hyperactivated YAP1, via manipulating multiple signaling pathways, induces epithelial cell transformation, MDSC (Myeloid-derived suppressor cells) accumulation, and pRCC development. Interestingly, we find that depletion of MDSC blocks YAP1-induced kidney overgrowth and tumorigenesis. Inhibiting YAP1 activity with MGH-CP1, a recently developed TEAD inhibitor, impedes MDSC accumulation and suppresses tumor development. Our results identify the disrupted Hippo/YAP signaling as a major contributor to pRCC and suggest that targeting the disrupted Hippo pathway represents a plausible strategy to prevent and treat pRCC.

Background Mucin 16 (MUC16) overexpression is linked with cancer progression, metastasis, and therapy resistance in high grade serous ovarian cancer and other malignancies. The cleavage of MUC16 forms independent bimodular fragments, the shed tandem repeat sequence which circulates as a protein bearing the ovarian cancer biomarker (CA125) and a proximal membrane-bound component which is critical in MUC16 oncogenic behavior. A humanized, high affinity antibody targeting the proximal ectodomain represents a potential therapeutic agent against MUC16 with lower antigenic potential and restricted human tissue expression. Results Here, we demonstrate the potential therapeutic versatility of the humanized antibody as a monoclonal antibody, antibody drug conjugate, and chimeric antigen receptor. We report the crystal structures of 4H11-scFv, derived from an antibody specifically targeting the MUC16 C-terminal region, alone and in complex with a 26-amino acid MUC16 segment resolved at 2.36 Å and 2.47 Å resolution, respectively. The scFv forms a robust interaction with an epitope consisting of two consecutive β-turns and a β-hairpin stabilized by 2 hydrogen bonds. The V H -V L interface within the 4H11-scFv is stabilized through an intricate network of 11 hydrogen bonds and a cation-π interaction. Conclusions Together, our studies offer insight into antibody-MUC16 ectodomain interaction and advance our ability to design agents with potentially improved therapeutic properties over anti-CA125 moiety antibodies.

Background Uterine serous cancer (USC) comprises around 10% of all uterine cancers. However, USC accounts for approximately 40% of uterine cancer deaths, which is attributed to tumor aggressiveness and limited effective treatment. Galectin 3 (Gal3) has been implicated in promoting aggressive features in some malignancies. However, Gal3’s role in promoting USC pathology is lacking. Methods We explored the relationship between LGALS3 levels and prognosis in USC patients using TCGA database, and examined the association between Gal3 levels in primary USC tumors and clinical-pathological features. CRISPR/Cas9-mediated Gal3-knockout (KO) and GB1107, inhibitor of Gal3, were employed to evaluate Gal3’s impact on cell function. Results TCGA analysis revealed a worse prognosis for USC patients with high LGALS3 . Patients with no-to-low Gal3 expression in primary tumors exhibited reduced clinical-pathological tumor progression. Gal3-KO and GB1107 reduced cell proliferation, stemness, adhesion, migration, and or invasion properties of USC lines. Furthermore, Gal3-positive conditioned media (CM) stimulated vascular tubal formation and branching and transition of fibroblast to cancer-associated fibroblast compared to Gal3-negative CM. Xenograft models emphasized the significance of Gal3 loss with fewer and smaller tumors compared to controls. Moreover, GB1107 impeded the growth of USC patient-derived organoids. Conclusion These findings suggest inhibiting Gal3 may benefit USC patients.

Abstract Background: Ovarian cancer (OC) is a lethal disease due to limited treatment options and the development of multidrug resistance. Bispecific T-cell engager (BiTE) therapy represents a promising immunotherapeutic approach to OC. Our previous findings suggest that resistance to BiTE therapy in OC is a complex interplay of factors including loss of tumor-associated antigens and epithelial-mesenchymal transition (EMT). Since the peritoneum is a relatively hypoxic (HX) tumor microenvironment, we hypothesized that HX plays a role in the modulation of MUC16 and potential resistance to BiTE therapy. Methods: After informed consent, samples were collected from patients with recurrent OC undergoing treatment with MUC16 BiTEs. For HX studies, we used OVCAR3 tumor cell lines which express full-length MUC16. Tumor cells were cultured under normoxic (NX) or HX conditions. The effect of HX on cell death was analyzed using an apoptosis assay. Cells were evaluated for changes in cell surface MUC16 expression by flow cytometry. MUC16 protein expression and EMT markers were analyzed by western blot. Proteasome inhibitors were used to stabilize MUC16 expression under HX conditions. Antibody internalization assays were conducted to assess MUC16 endocytosis and recycling. Results: In patients who progressed on MUC16 BiTEs, we found decreased MUC16 expression on tumor samples isolated from ascites samples. Using OVCAR3 cells, we found significantly decreased MUC16 expression on ovarian cancer cells grown under HX conditions (Fold change: 0.4 vs NX, p<0.001). Flow cytometry analysis revealed a time-dependent loss of MUC16 expression on the cell surface under HX (64.22% after 9 days HX vs 88.26% NX), highlighting the susceptibility of MUC16 to HX-induced downregulation. Treatment with proteasome inhibitors partially rescued MUC16 expression under HX conditions (Fold change: 1.4 vs NX, p<0.001). Notably, HX did not induce necrotic cell death in these tumor cells. In addition to the loss of MUC16, tumor cells under HX conditions showed alterations in EMT markers. E-cadherin was downregulated with prolonged HX exposure (Fold change: 0.3 vs NX, p < 0.001), while Vimentin (Fold change: 7.1 vs NX, p < 0.001) and Snail (Fold change: 2.8 vs NX, p < 0.001) were upregulated, indicating a potential shift towards a more aggressive phenotype. Epidermal growth factor receptor (EGFR) was also upregulated (Fold change: 4.9 vs NX, p < 0.001), potentially contributing to enhanced survival and invasion of OC cells under HX stress. Finally, under HX conditions, MUC16 internalization was slower in response to antibody binding over time due to reduced cell antigen expression, corroborating the observed MUC16 loss. Conclusion: Understanding hypoxia-induced MUC16 downregulation and its downstream effects on EMT markers and CA125 expression provides crucial insight into the mechanisms of BiTE therapy resistance in ovarian cancer. Citation Format: Mengyao Xu, Syem K. Barakzai, Artem Kononenko, Raj Kumar, Irva Veillard, Eugene Kim, Timothy S. Bond, David R. Spriggs, Bo R. Rueda, Oladapo Yeku. Hypoxia-induced downregulation of MUC16: A key player in bispecific T-cell engager resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7557.

Supplementary Figure from Dual Fluorescence Isogenic Synthetic Lethal Kinase Screen and High-Content Secondary Screening for MUC16/CA125-Selective Agents