Proceedings of the National Academy of Sciences

Defining CDK12 as a tumor suppressor and therapeutic target in mouse models of tubo-ovarian high-grade serous carcinoma

Ovarian cancer is the sixth leading cause of cancer death among American women, with most fatalities attributable to tubo-ovarian high-grade serous carcinoma (HGSC). This malignancy usually develops resistance to conventional chemotherapy, underscoring the need for robust preclinical models to guide the development of novel therapies. Here, we introduce an HGSC mouse model generated via Ovgp1 -driven Cre recombinase effecting CRISPR/Cas9-mediated deletion of Trp53, Rb1 , and Nf1 tumor suppressors in mouse oviductal epithelium ( m-sgPRN model). Cyclin-dependent kinase 12 (CDK12) inactivation—frequently observed in human HGSC—is associated with poorer outcomes, DNA damage accumulation (including tandem duplications), and increased tumor immunogenicity. In our system, coablation of Cdk12 ( m-sgPRN;Cdk12KO ) recapitulated hallmark features of HGSC, while accelerating tumor progression and reducing survival. In a conventional (Cre-lox-mediated) Trp53/Nf1/Rb1 triple knockout model with concurrent Cdk12 ablation ( PRN ; Cdk12KO mice), we observed T cell–rich immune infiltrates mirroring those seen clinically. We established both models as subcutaneous or intraperitoneal syngeneic allografts of CDK12 -inactivated HGSC that exhibited sensitivity to immune checkpoint blockade. Furthermore, a CRISPR/Cas9 synthetic lethality screen in PRN;Cdk12KO -derived cell lines identified CDK13—an essential paralog of CDK12—as the most depleted candidate, confirming a previously reported synthetic lethal interaction. Pharmacologic CDK13/12 degradation (employing YJ1206) demonstrated enhanced efficacy in cell lines derived from both m-sgPRN;Cdk12KO and PRN ; Cdk12KO models. Our results define CDK12 as a key tumor suppressor in tubo-ovarian HGSC and highlight CDK13 targeting as a promising therapeutic approach in CDK12 -inactive disease. Additionally, we have established valuable in vivo resources to facilitate further investigation and drug development in this challenging malignancy.

CRISPR screen reveals a simultaneous targeted mechanism to reduce cancer cell selenium and increase lipid oxidation to induce ferroptosis

Ferroptosis is a cell death mechanism distinguished by its dependence on iron-mediated lipid oxidation. Cancer cells highly resistant to conventional therapies often demonstrate lipid metabolic and redox vulnerabilities that sensitize them to cell death by ferroptosis. These include a unique dependency on the lipid antioxidant selenoenzyme, glutathione peroxidase 4 (GPx4), that acts as a ferroptosis inhibitor. Synthetic high-density lipoprotein-like nanoparticle (HDL NP) targets the high-affinity HDL receptor scavenger receptor class B type 1 (SR-B1) and regulates cell and cell membrane lipid metabolism. Recently, we reported that targeting cancer cell SR-B1 with HDL NP depleted cell GPx4, which is accompanied by increased cell membrane lipid peroxidation and cancer cell death. These data suggest that HDL NP may induce ferroptosis. Thus, we conducted an unbiased CRISPR-based positive selection screen and target validation studies in ovarian clear cell carcinoma (OCCC) cell lines to ascertain the mechanism through which HDL NP regulates GPx4 and kills cancer cells. The screen revealed two genes, acyl-CoA synthetase long chain family member 4 (ACSL4) and thioredoxin reductase 1 (TXNRD1), whose loss conferred resistance to HDL NP. Validation of ACSL4 supports that HDL NP induces ferroptosis as the predominant mechanism of cell death, while validation of TXNRD1 revealed that HDL NP reduces cellular selenium and selenoprotein production, most notably, GPx4. Accordingly, we define cancer cell metabolic targets that can be simultaneously actuated by a multifunctional, synthetic HDL NP ligand of SR-B1 to kill cancer cells by ferroptosis.

Triple checkpoint blockade of PD-1, Tim-3, and Lag-3 enhances adoptive T cell immunotherapy in a mouse model of ovarian cancer

The five-year survival rate for ovarian cancer patients remains below 50%, underscoring the need for innovative therapies. One promising approach involves engineering T cells to specifically target proteins uniquely overexpressed in tumors, thereby controlling tumor growth without toxicity to healthy tissues. Mesothelin (MSLN) contributes to the malignant and invasive phenotype in ovarian cancer and has limited expression in healthy cells, making it a candidate immunotherapy target. Our previous results in a mouse model of ovarian cancer demonstrated that T cells engineered to express a T cell receptor (TCR) targeting MSLN (TCR MSLN ) mediated therapeutic activity, delaying tumor growth and prolonging mouse survival. However, inhibitory ligands expressed in the tumor microenvironment (TME) interacted with inhibitory receptors on activated T cells, suppressing antitumor function. We hypothesized combining engineered T cells with checkpoint blockade would enhance T cell function and improve therapeutic efficacy, but administration of monospecific antibodies targeting individual inhibitory pathways had no significant impact on T cell efficacy. By contrast, the combination of PD-1, Tim-3, and Lag-3 blockade with engineered T cells significantly improved T cell function and overall animal survival relative to treatment with antibody alone or TCR MSLN with singlet or doublet antibody combinations. Single-cell RNA sequencing revealed TCR MSLN T cells treated with the triplet antibody combination increased expression of genes involved in interferon responses and metabolic function, and reduced expression of genes associated with exhaustion. These results suggest that strategies to disrupt multiple inhibitory pathways simultaneously may be necessary for improved adoptive T cell therapy efficacy in patients.

Integrated mutational landscape analysis of endometrial stromal sarcoma

Endometrial stromal sarcoma (ESS) is a rare uterine malignancy with limited treatment options. We performed integrated whole-genome, whole-exome, and transcriptome sequencing on 80 ESS tumors, comprising 32 low-grade (LG) and 48 high-grade (HG) tumors, to characterize their genetic landscape. The overall mutation burden was modest, with no significant difference between grades; however, we identified six hypermutated cases (7.5%) harboring POLE or mismatch repair mutations, genomic features predictive of immunotherapy response. We identified focal RAD54B amplifications in 15 tumors (18.8%), leading to elevated RAD54B expression and significantly shorter survival. This establishes RAD54B as an oncogenic driver in ESS. Known tumor suppressors (PTEN, TP53) were frequently mutated in HG-ESS but rare in LG-ESS, highlighting distinct grade-specific drivers of malignancy. HG-ESS exhibited widespread chromosomal gains, frequent loss of cell-cycle regulators (RB1, CDKN2A), and numerous private gene fusions arising from complex DNA rearrangements. In contrast, LG-ESS were defined by canonical fusions (e.g., JAZF1–SUZ12 ) and co-occurring deletions in metabolic regulator genes (TSC2, STK11). Finally, in an activating NRAS-mutant (p.Q61R) HG-ESS xenograft, the combination of MEK and FAK inhibition dramatically suppressed tumor growth and prolonged survival, highlighting a promising targeted treatment strategy. Overall, our comprehensive analysis defines the molecular basis of ESS and provides a strong preclinical rationale for precision therapies in this aggressive cancer.

Leveraging chromatin packing domains to target chemoevasion in vivo

Cancer cells exhibit a remarkable resilience to cytotoxic stress, often adapting through transcriptional changes linked to alterations in chromatin structure. In several types of cancer, these adaptations involve epigenetic modifications and restructuring of topologically associating domains. However, the underlying principles by which chromatin architecture facilitates such adaptability across different cancers remain poorly understood. To investigate the role of chromatin in this process, we developed a physics-based model that connects chromatin organization to cell fate decisions, such as survival following chemotherapy. Our model builds on the observation that chromatin forms packing domains, which influence transcriptional activity through macromolecular crowding. The model accurately predicts chemoevasion in vitro, suggesting that changes in packing domains affect the likelihood of survival. Consistent results across diverse cancer types indicate that the model captures fundamental principles of chromatin-mediated adaptation, independent of the specific cancer or chemotherapy mechanisms involved. Based on these insights, we hypothesized that compounds capable of modulating packing domains, termed Transcriptional Plasticity Regulators (TPRs), could prevent cellular adaptation to chemotherapy. We conducted a proof-of-concept compound screen using live-cell chromatin imaging to identify several TPRs that synergistically enhanced chemotherapy-induced cell death. The most effective TPR significantly improved therapeutic outcomes in a patient-derived xenograft model of ovarian cancer. These findings underscore the central role of chromatin in cellular adaptation to cytotoxic stress and present a framework for enhancing cancer therapies, with broad potential across multiple cancer types.

Loss of XIST lncRNA unlocks stemness and cellular plasticity in ovarian cancer

Plasticity, a key hallmark of cancer, enables cells to transition into different states, driving tumor heterogeneity. This cellular plasticity is associated with cancer progression, treatment resistance, and relapse. Cancer stem cells (CSCs) play a central role in this process, yet the molecular factors underlying cancer cell stemness remain poorly understood. In this study, we explored the role of XIST (X-inactive specific transcript) long noncoding RNA in ovarian cancer stemness and plasticity through in silico and in vitro analyses. We found that XIST is significantly down-regulated in ovarian tumors, with low XIST expression linked to a higher stemness index and lower overall survival. Knocking down XIST in ovarian cancer cells enhanced stemness, particularly increasing mesenchymal-like CSCs, and under hypoxic conditions, it promoted epithelial-like CSC markers. Our findings suggest that XIST loss leads to CSC enrichment and cellular plasticity in ovarian cancer, pointing to potential therapeutic targets for patients with low XIST expression.

PARG inhibitor sensitivity correlates with accumulation of single-stranded DNA gaps in preclinical models of ovarian cancer

Poly (ADP-ribose) glycohydrolase (PARG) is a dePARylating enzyme which promotes DNA repair by removal of poly (ADP-ribose) (PAR) from PARylated proteins. Loss or inhibition of PARG results in replication stress and sensitizes cancer cells to DNA-damaging agents. PARG inhibitors are now undergoing clinical development for patients having tumors with homologous recombination deficiency (HRD), such as cancer patients with germline or somaticBRCA1/2-mutations. PARP inhibitors kill BRCA-deficient cancer cells by increasing single-stranded DNA gaps (ssGAPs) during replication. Here, we report that, like PARP inhibitor (PARPi), PARG inhibitor (PARGi) treatment also causes an accumulation of ssGAPs in sensitive cells. PARGi exposure increased accumulation of S-phase-specific PAR, a marker for Okazaki fragment processing (OFP) defects on lagging strands and induced ssGAPs, in sensitive cells but not in resistant cells. PARGi also caused accumulation of PAR at the replication forks and at the ssDNA sites in sensitive cells. Additionally, PARGi exhibited monotherapy activity in specific HR-deficient, as well as HR-proficient, patient-derived, or patient-derived xenograft (PDX)-derived organoids of ovarian cancer, and drug sensitivity directly correlated with the accumulation of ssGAPs. Taken together, PARGi treatment results in toxic accumulation of PAR at replication forks resulting in ssGAPs due to OFP defects during replication. Regardless of theBRCA/HRD-status, the induction of ssGAPs in preclinical models of ovarian cancer cells correlates with PARGi sensitivity. Patient-derived organoids (PDOs) may be a useful model system for testing PARGi sensitivity and functional biomarkers.

OTUB2 silencing promotes ovarian cancer via mitochondrial metabolic reprogramming and can be synthetically targeted by CA9 inhibition

Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel–Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment ofOTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.

Targeting and monitoring ovarian cancer invasion with an RNAi and peptide delivery system

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.

Tumor resistance to anti-mesothelin CAR-T cells caused by binding to shed mesothelin is overcome by targeting a juxtamembrane epitope

Despite many clinical trials, CAR-T cells are not yet approved for human solid tumor therapy. One popular target is mesothelin (MSLN) which is highly expressed on the surface of about 30% of cancers including mesothelioma and cancers of the ovary, pancreas, and lung. MSLN is shed by proteases that cleave near the C terminus, leaving a short peptide attached to the cell. Most anti-MSLN antibodies bind to shed MSLN, which can prevent their binding to target cells. To overcome this limitation, we developed an antibody (15B6) that binds next to the membrane at the protease-sensitive region, does not bind to shed MSLN, and makes CAR-T cells that have much higher anti-tumor activity than a CAR-T that binds to shed MSLN. We have now humanized the Fv (h15B6), so the CAR-T can be used to treat patients and show that h15B6 CAR-T produces complete regressions in a hard-to-treat pancreatic cancer patient derived xenograft model, whereas CAR-T targeting a shed epitope (SS1) have no anti-tumor activity. In these pancreatic cancers, the h15B6 CAR-T replicates and replaces the cancer cells, whereas there are no CAR-T cells in the tumors receiving SS1 CAR-T. To determine the mechanism accounting for high activity, we used an OVCAR-8 intraperitoneal model to show that poorly active SS1-CAR-T cells are bound to shed MSLN, whereas highly active h15B6 CAR-T do not contain bound MSLN enabling them to bind to and kill cancer cells.

Hyperactive Rac stimulates cannibalism of living target cells and enhances CAR-M-mediated cancer cell killing

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62Kalso causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62Kstimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62Kbone-marrow-derived macrophages also cannibalize primary Rac2+/E62KT cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62Kmacrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62Kalso enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62Khuman immunodeficiency and enhance CAR-M cancer immunotherapy.

Lipidomic profiling of endometrial cancer using desorption electrospray ionization mass spectrometry imaging

Novel technologies are required to improve endometrial cancer diagnosis and enhance the early detection of preinvasive precursors. Herein, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) was used to differentiate malignant from benign endometrial tissues and assess lipidomic differences among patients with obesity and diabetes. Reverse phase protein array (RPPA) analysis was performed in the same patients to gain insights into the altered signaling pathways and investigate the protein expression levels in endometrial cancer cases and controls. Tissues from 64 women (50 cancer, 14 benign) were analyzed with DESI-MSI achieving 90% sensitivity and 93% specificity. Discriminatory spectral features were primarily phospholipids [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)], all elevated in cancer. Proteomics revealed upregulated proteins linked to commonly dysregulated pathways in endometrial cancer, namely PI3K/AKT/mTOR, MAPK/RAS, and Wnt signaling pathways. Lipidomic differences were found between high- (obesity/diabetes) and low-risk phenotypes (no obesity/diabetes), with PE and PS elevated in high-risk benign and PE reduced in high-risk cancer cases. A single phospholipid (PE(O-38:4)) was found as discriminatory in both normal and cancer cohorts, which may serve as biomarker in women with benign histology at high-risk of developing endometrial cancer. This study reports the application of DESI-MSI for lipidomic characterisation of endometrial cancer.

Uterine organoids reveal insights into epithelial specification and plasticity in development and disease

Understanding how epithelial cells in the female reproductive tract (FRT) differentiate is crucial for reproductive health, yet the underlying mechanisms remain poorly defined. At birth, FRT epithelium is highly malleable, allowing differentiation into various epithelial types, but the regulatory pathways guiding these early cell fate decisions are unclear. Here, we use neonatal mouse endometrial organoids and assembloid coculture models to investigate how innate cellular plasticity and external mesenchymal signals influence epithelial differentiation. Our findings demonstrate that uterine epithelium undergoes marked age-dependent changes, transitioning from a highly plastic state capable of forming both monolayered and multilayered structures to a more restricted fate as development progresses. Interestingly, parallels emerge between the developmental plasticity of neonatal uterine epithelium and pathological conditions such as endometrial cancer, where similar regulatory mechanisms may reactivate, driving abnormal epithelial differentiation and tumorigenesis. These results not only deepen our understanding of early uterine development but also offer a valuable model for studying the progression of reproductive diseases and cancers.

Modulation of the PGRMC1/NLRP7/HLA-C axis by autophagy is linked to both spontaneous preterm birth and gestational choriocarcinoma

Spontaneous preterm birth (SPTB) and gestational choriocarcinoma are both associated with complex physiological processes that significantly impact maternal health. While the molecular mechanisms underlying SPTB and gestational choriocarcinoma remain poorly understood, emerging evidence suggests that immune regulation plays a crucial role in both conditions. In this study, we revealed that progesterone regulates autophagy via the noncanonical progesterone receptor membrane component 1 (PGRMC1), which then modulates NLRP7 levels, thereby impacting HLA-C expression in the JEG3 cell line, an extravillous trophoblast (EVT) model. Furthermore, a significant positive correlation between NLRP7 and HLA-C expression was observed in EVTs from placental tissues and choriocarcinoma samples. In cases of SPTB, we found both reduced expression of NLRP7 and HLA-C in EVTs. Similarly, in gestational choriocarcinoma samples, we observed significantly lower expression levels of NLRP7 and HLA-C, further suggesting a shared immune evasion mechanism. These findings not only provide insights into the molecular mechanisms underlying both SPTB and choriocarcinoma but also identify the progesterone-driven NLRP7/HLA-C axis as a promising target for therapeutic intervention, offering strategies for improving outcomes in both conditions.

Loss of ICOSL expression in the progression to cervical carcinoma

Human papillomavirus (HPV)–related lesions contain types with benign outcomes and those with a risk of progression to cancer. We addressed the role of immune surveillance in 76 cervical biopsies (normal = 23, HPV+ benign = 16, HPV+ precancer = 37) by studying the infiltration of cytotoxic T cells and the expression of the immune modulators PDL1, ICOSL, and miR-155 and compared the data to 101 cervical squamous cell carcinomas. In the normal cervix, ICOSL expression was restricted to the endocervical epithelia whereas neither miR-155 nor PDL1 were detected. MiR-155 was up-regulated in both the benign (88%) and precancerous (92%) HPV squamous intraepithelial lesions (SIL) and colocalized to cells in the upper part of the lesion that is the area with productive viral infection. Both PDL1 (95%) and ICOSL (89%) were only evident in the precancerous SIL and each localized to squamous cells in the basal aspect that lacked replicating virus. In both microinvasive and invasive cervical squamous cell cancer miR-155 expression remained high (83%) as did PDL1 expression (80%) but ICOSL detection was reduced to 17%. Infiltration by CD8+ T cells was intense in the invasive lesions and these cells were mostly inactive as determined by the lack of granzyme B colocalization. It is concluded that miR-155 expression is a marker of HPV infection in both benign and precancerous lesions, whereas the approximately 10% of the latter lesions that progress to cancer gain PDL1 and lose ICOSL expression, which are important factors in avoiding immune surveillance.

Contextualizing racial associations in gene expression in patients with uterine serous carcinoma

Computational modeling of ovarian cancer dynamics suggests optimal strategies for therapy and screening

Significance The optimal timing of surgery/chemotherapy and the benefits of earlier diagnosis of HGSC remain controversial. We developed a mathematical framework of tumor dynamics, populated the model with primary clinical data, and reliably recapitulated clinical observations. Our model predicts that 1) PDS is superior to NACT with relatively small tumor burden and when complete debulking is feasible, 2) timely adjuvant chemotherapy is critical for the outcome of PDS with <1-mm residual tumors, 3) earlier detection of relapse is unlikely beneficial with current therapies, and 4) earlier detection of primary HGSC could have substantial benefit. These results provide insights into the evolutionary dynamics of HGSC, argue for new clinical trials to optimize therapy, and are potentially applicable to other tumor types.

ACTL6A promotes repair of cisplatin-induced DNA damage, a new mechanism of platinum resistance in cancer

Significance Platinum resistance remains as a major issue in the therapy for many types of cancer. However, the mechanisms of resistance have not been fully elucidated. ACTL6A gene is frequently amplified in several types of cancer such as lung squamous cell carcinoma, ovarian cancer, and esophageal cancer. ACTL6A is a subunit shared by multiple complexes, including SWI/SNF, INO80, and NuA4/TIP60. We unveil a new role for ACTL6A in repairing cisplatin-induced DNA damage, providing a novel mechanism for cisplatin resistance. We also show that the action of ACTL6A in the repair of cisplatin-induced DNA lesions is through the SWI/SNF remodeling complex. Furthermore, we demonstrate that an HDAC inhibitor can abolish cisplatin resistance caused by ACTL6A overexpression.

Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids

Fatty acids are an important source of energy and a key component of phospholipids in membranes and organelles. Saturated fatty acids (SFAs) are converted into unsaturated fatty acids (UFAs) by stearoyl Co-A desaturase (SCD), an enzyme active in cancer. Here, we studied how the dynamics between SFAs and UFAs regulated by SCD impacts ovarian cancer cell survival and tumor progression. SCD depletion or inhibition caused lower levels of UFAs vs. SFAs and altered fatty acyl chain plasticity, as demonstrated by lipidomics and stimulated Raman scattering (SRS) microscopy. Further, increased levels of SFAs resulting from SCD knockdown triggered endoplasmic reticulum (ER) stress response with brisk activation of IRE1α/XBP1 and PERK/eIF2α/ATF4 axes. Disorganized ER membrane was visualized by electron microscopy and SRS imaging in ovarian cancer cells in which SCD was knocked down. The induction of long-term mild ER stress or short-time severe ER stress by the increased levels of SFAs and loss of UFAs led to cell death. However, ER stress and apoptosis could be readily rescued by supplementation with UFAs and reequilibration of SFA/UFA levels. The effects of SCD knockdown or inhibition observed in vitro translated into suppression of intraperitoneal tumor growth in ovarian cancer xenograft models. Furthermore, a combined intervention using an SCD inhibitor and an SFA-enriched diet initiated ER stress in tumors growing in vivo and potently blocked their dissemination. In all, our data support SCD as a key regulator of the cancer cell fate under metabolic stress and point to treatment strategies targeting the lipid balance.

Treatment of ovarian cancer with modified anthrax toxin

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

Tumor FAK orchestrates immunosuppression in ovarian cancer via the CD155/TIGIT axis

Significance High-grade serous ovarian carcinoma (HGSOC) is an immunotherapy-resistant lethal cancer. An HGSOC hallmark is elevated checkpoint pathway ligand expression that limits antitumor immune responses. Computational, preclinical, and patient tumor multiplexed analyses revealed that tumor-associated focal adhesion kinase (FAK) activation regulates CD155 expression, a checkpoint ligand for TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains). Using an aggressive mouse ovarian tumor model, we find that combined oral FAK inhibitor plus function-blocking TIGIT antibody immunotherapy reduced tumor burden, prolonged mouse survival, and led to immune cell activation and tertiary lymphoid structure formation, hallmarks of an antitumor immune response. As FAK is commonly overexpressed in HGSOC tumors, targeting FAK and TIGIT may limit tumor immune evasion.

Senescence induction dictates response to chemo- and immunotherapy in preclinical models of ovarian cancer

Significance Efforts to understand and find new treatment options for high-grade serous ovarian cancer (HGSOC) have been confounded by a paucity of immune-competent models that accurately reflect the genetics and biology of the disease. Here, we leverage somatic tissue engineering to develop a fast and flexible immune-competent mouse model of HGSOC and reveal mechanistic insights into factors that dictate the response of ovarian tumors to conventional chemotherapy and immune checkpoint blockade. Our results identify a genotype-dependent therapy-induced senescence program that mediates sensitivity and resistance to first line chemotherapy and point to strategies to harness the senescence program to sensitize ovarian tumors to immune checkpoint blockade.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1

Significance Persistent infections of oncogenic HPVs can lead to cervical, anogenital, and head-and-neck cancers. We report that HPV-infected cervical lesions display aberrant expression of 194 long-noncoding RNAs, including lnc-FANCI-2 . We show that, in cell and tissue cultures, lnc-FANCI-2 is induced primarily by the oncogenic HPV E7 protein via interacting with a cellular transcription factor YY1. HPV infection also increases YY1 protein expression by reducing miR-29a, which targets the 3′ UTR of YY1 RNA. In vivo and in vitro, the nearby DNA repair gene FANCI is co-upregulated. These findings highlight an aspect on how oncogenic HPV E7 contributes to pathogenesis.

PPT1 is a negative regulator of STING signaling in cancer cells and its inhibition reactivates immune surveillance in cold tumors

Immunotherapy modalities have revolutionized cancer treatment for a number of metastatic and treatment-refractory tumor types. Still, many malignancies that lack T cell infiltration and are termed immunologically “cold” fail to respond to these modalities. One approach to increase tumor immunogenicity has been to induce stimulator of interferon gene (STING) and downstream interferon signaling that is often dysregulated in cold tumors. Despite some early success of STING agonists in preclinical cancer models, these approaches have not been successful in the clinic due to poor tumor penetrance and systemic toxicities. Here, we performed a genome-wide CRISPR screen to uncover therapeutic targets to activate STING expression in human tumors. We identified the lysosomal hydrolase Palmitoyl Protein Thioesterase1 (PPT1) as a negative regulator of STING highly expressed in cold ovarian and prostate tumors. Genetic or pharmacological PPT1 suppression increased STING protein stability and its downstream activation of interferon and inflammatory cytokine signaling to enhance T cell migration. Treatment of preclinical prostate and ovarian cancer models expressing low levels of STING with the small molecule PPT1 inhibitor GNS561 enhanced STING expression and activation, leading to infiltration and activation of cytotoxic T cells that turned these tumors “hot” and reduced tumor growth, fibrosis, and dissemination without toxicity. Further analysis demonstrated that PPT1 is associated with reduced STING expression, CD8 + T cell numbers, overall survival, and immunotherapy outcomes in ovarian and prostate cancer patients. Thus, PPT1 inhibition may be a promising approach to activate STING and potentiate the effects of immunotherapy in cold tumors.

Single-nuclei sequencing of uterine serous carcinoma reveals racial differences in immune signaling

Significant racial disparities exist between Black and White patients with uterine serous carcinoma (USC). While the reasons for these disparities are unclear, several studies have demonstrated significantly different rates of driver mutations between racial groups, including TP53. However, limited research has investigated the transcriptional differences of tumors or the composition of the tumor microenvironment (TME) between these groups. Here, we report the single-nuclei RNA-sequencing profiles of primary USC tumors from diverse racial backgrounds. We find that there are significant differences between the tumors of Black and White patients. Tumors from Black patients exhibited higher expression of specific genes associated with aggressiveness, such as PAX8, and axon guidance and synaptic signaling pathways. We also demonstrated that T cell populations are reduced in the tumor tissue compared to matched benign, while anti-inflammatory macrophage populations are retained within the TME. Furthermore, we investigated the connection between PAX8 overexpression and immunosuppression in USC through regulation of several cytokines and chemokines. Notably, we show that PAX8 activity can influence macrophage gene expression and protein secretion. These studies provide a detailed understanding of the USC transcriptome and TME, and identify differences in tumor biology from patients of different racial backgrounds.

DNA2 protein destruction dictates DNA hyperexcision, cGAS–STING activation, and innate immune response in CDK12-deregulated cancers

CDK12 primarily functions as a transcription regulatory cyclin-dependent kinase (CDK) that controls mRNA elongation, splicing, and polyadenylation. The CDK12 gene is implicated in human cancers since it is frequently mutated and/or deleted in prostate and ovarian cancer but paradoxically amplified in breast cancer. Here, we demonstrate that CDK12 promotes serine-933 phosphorylation of DNA2, a nuclease/helicase critical for replication fork stress regulation, and the phosphorylation subsequently facilitates DNA2 polyubiquitination and degradation mediated by the APC/C CDC20 E3 ubiquitin ligase. CDK12 inactivation induces but amplification suppresses genome-wide expression of interferon response and antigen processing and presentation machinery genes in ovarian and breast cancer cells, respectively. Besides causing aberrant DNA2 stabilization, replication stress, genomic instability, and cytosolic double-stranded DNA (dsDNA) accumulation, CDK12 loss also triggers cGAS–STING activation and innate immune response, which can be reversed by forced expression of replication protein A (RPA) subunits or DNA2 depletion. Our findings identify DNA2 as a phosphorylation substrate of CDK12, connecting CDK12 to cell cycle regulation. These data also reveal DNA2 protein destruction as a critical mechanism that dictates genomic instability, cGAS–STING signaling activation, and innate immune response in CDK12-deregulated cancers.

Harald zur Hausen (1936 to 2023): Discoverer of human papillomavirus infection as the main cause of cervical cancer

Reply to Rollin et al.: Clarifying the multifactorial origins of racial disparities in uterine serous carcinoma

Integrated mutational landscape analysis of poorly differentiated high-grade neuroendocrine carcinoma of the uterine cervix

High-grade neuroendocrine cervical cancers (NETc) are exceedingly rare, highly aggressive tumors. We analyzed 64 NETc tumor samples by whole-exome sequencing (WES). Human papillomavirus DNA was detected in 65.6% (42/64) of the tumors. Recurrent mutations were identified in PIK3CA, KMT2D/MLL2, K-RAS, ARID1A, NOTCH2, and RPL10. The top mutated genes included RB1, ARID1A, PTEN, KMT2D / MLL2, and WDFY3, a gene not yet implicated in NETc. Somatic CNV analysis identified two copy number gains (3q27.1 and 19q13.12) and five copy number losses (1p36.21/5q31.3/6p22.2/9q21.11/11p15.5). Also, gene fusions affecting the ACLY-CRHR1 and PVT1-MYC genes were identified in one of the eight samples subjected to RNA sequencing. To resolve evolutionary history, multiregion WES in NETc admixed with adenocarcinoma cells was performed (i.e., mixed-NETc). Phylogenetic analysis of mixed-NETc demonstrated that adenocarcinoma and neuroendocrine elements derive from a common precursor with mutations typical of adenocarcinomas. Over one-third (22/64) of NETc demonstrated a mutator phenotype of C > T at CpG consistent with deficiencies in MBD4 , a member of the base excision repair (BER) pathway. Mutations in the PI3K/AMPK pathways were identified in 49/64 samples. We used two patient-derived-xenografts (PDX) (i.e., NET19 and NET21) to evaluate the activity of pan-HER (afatinib), PIK3CA (copanlisib), and ATR (elimusertib) inhibitors, alone and in combination. PDXs harboring alterations in the ERBB2/PI3K/AKT/mTOR/ATR pathway were sensitive to afatinib, copanlisib, and elimusertib ( P < 0.001 vs. controls). However, combinations of copanlisib/afatinib and copanlisib/elimusertib were significantly more effective in controlling NETc tumor growth. These findings define the genetic landscape of NETc and suggest that a large subset of these highly lethal malignancies might benefit from existing targeted therapies.

Homologous recombination–deficient mutation cluster in tumor suppressor RAD51C identified by comprehensive analysis of cancer variants

Mutations in homologous recombination (HR) genes, including BRCA1 , BRCA2 , and the RAD51 paralog RAD51C , predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs. A cluster of mutations was identified in and around the Walker A box that led to impairments in HR, interactions with three other RAD51 paralogs, binding to single-stranded DNA, and ATP hydrolysis. We generated structural models of the two RAD51 paralog complexes containing RAD51C, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3. Together with our functional and biochemical analyses, the structural models predict ATP binding at the interface of RAD51C interactions with other RAD51 paralogs, similar to interactions between monomers in RAD51 filaments, and explain the failure of RAD51C variants in binding multiple paralogs. Ovarian cancer patients with variants in this cluster showed exceptionally long survival, which may be relevant to the reversion potential of the variants. This comprehensive analysis provides a framework for RAD51C variant classification. Importantly, it also provides insight into the functioning of the RAD51 paralog complexes.

Discovery of antibodies and cognate surface targets for ovarian cancer by surface profiling

Although antibodies targeting specific tumor-expressed antigens are the standard of care for some cancers, the identification of cancer-specific targets amenable to antibody binding has remained a bottleneck in development of new therapeutics. To overcome this challenge, we developed a high-throughput platform that allows for the unbiased, simultaneous discovery of antibodies and targets based on phenotypic binding profiles. Applying this platform to ovarian cancer, we identified a wide diversity of cancer targets including receptor tyrosine kinases, adhesion and migration proteins, proteases and proteins regulating angiogenesis in a single round of screening using genomics, flow cytometry, and mass spectrometry. In particular, we identified BCAM as a promising candidate for targeted therapy in high-grade serous ovarian cancers. More generally, this approach provides a rapid and flexible framework to identify cancer targets and antibodies.

Sulfated glycosaminoglycans mediate prion-like behavior of p53 aggregates

Significance Approximately 50 human diseases are associated with deposition of abnormally aggregated proteins that propagate in a prion-like manner. The tumor suppressor p53 forms amyloid-like aggregates; however, how p53 aggregates propagate remains unclear. Here, we identified heparan sulfate (HS), the common and major nonprotein component of in vivo deposits of various protein aggregates, as a mediator of prion-like propagation of p53 aggregates in cultured cells. Our immunohistochemical analysis showing codeposition of p53 and highly sulfated domains of HS in ovarian cancer tissues strongly support the role of HS-mediated propagation of p53 aggregates in cancer pathology. Accordingly, our results provide a mechanism of propagation of p53 aggregates that is mediated by HS. Elucidation of detailed biological relevance in cancer is warranted.

Targeting progesterone signaling prevents metastatic ovarian cancer

SignificanceWhy women carrying a pathogenic germlineBRCA1mutation are predisposed to ovarian and breast cancer remains elusive. This study points to ovarian progesterone as a culprit. Generally,BRCA1-mutation carriers exhibit high yet individually varying levels of progesterone during the menstrual cycle. Although not allBRCA1-mutation carriers develop these cancers, all of them are advised to undergo prophylactic surgeries at a young age (under 40 y to 45 y) to prevent ovarian and breast cancer. Insights from robust in vivo findings in this study offer a novel concept: Targeting progesterone signaling with antiprogestins could be an effective nonsurgical prophylactic option for ovarian and breast cancer prevention for these high-risk women.

An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins

Significance Researchers and clinicians rely on robust staining methods to detect and visualize biomolecules of interest. Mucins are heavily glycosylated proteins expressed on cells throughout the human body that have historically proved challenging to stain with high specificity, hindering attempts to explore their roles in health and disease. Here, we address this limitation using catalytically inactivated mucin-cleaving bacterial proteases as staining reagents. The differing specificities of these enzymes are retained in their binding properties, enabling a new depth in the analysis of mucins on cells and patient tissues.

Specific targeting of ovarian tumor-associated macrophages by large, anionic nanoparticles

Significance Tumor-associated macrophages (TAMs) are abundant in tumor microenvironments and are predominantly immunosuppressive. The ratio between immunosuppressive M2 TAMs and proinflammatory M1 TAMs correlates with worse outcomes for many cancers, including ovarian cancer. TAMs are potential targets for immunotherapy, and here we show that, when relatively large (>100-nm) anionic nanoparticles are administered intraperitoneally (i.p.), they selectively accumulate in TAMs with high efficiency (>60% of injected dose). The composition of particles that achieve this targeting appears to be quite flexible. Thus, based on our results, many formulations can now be tested for targeted immunotherapy of ovarian cancer and other cancers of the i.p. cavity.

The intelligent knife (iKnife) and its intraoperative diagnostic advantage for the treatment of cervical disease

Clearance of surgical margins in cervical cancer prevents the need for adjuvant chemoradiation and allows fertility preservation. In this study, we determined the capacity of the rapid evaporative ionization mass spectrometry (REIMS), also known as intelligent knife (iKnife), to discriminate between healthy, preinvasive, and invasive cervical tissue. Cervical tissue samples were collected from women with healthy, human papilloma virus (HPV) ± cervical intraepithelial neoplasia (CIN), or cervical cancer. A handheld diathermy device generated surgical aerosol, which was transferred into a mass spectrometer for subsequent chemical analysis. Combination of principal component and linear discriminant analysis and least absolute shrinkage and selection operator was employed to study the spectral differences between groups. Significance of discriminatory m/z features was tested using univariate statistics and tandem MS performed to elucidate the structure of the significant peaks allowing separation of the two classes. We analyzed 87 samples (normal = 16, HPV ± CIN = 50, cancer = 21 patients). The iKnife discriminated with 100% accuracy normal (100%) vs. HPV ± CIN (100%) vs. cancer (100%) when compared to histology as the gold standard. When comparing normal vs. cancer samples, the accuracy was 100% with a sensitivity of 100% (95% CI 83.9 to 100) and specificity 100% (79.4 to 100). Univariate analysis revealed significant MS peaks in the cancer-to-normal separation belonging to various classes of complex lipids. The iKnife discriminates healthy from premalignant and invasive cervical lesions with high accuracy and can improve oncological outcomes and fertility preservation of women treated surgically for cervical cancer. Larger in vivo research cohorts are required to validate these findings.

DPF2 reads histone lactylation to drive transcription and tumorigenesis

Lysine lactylation (Kla) is a new type of histone mark implicated in the regulation of various functional processes such as transcription. However, how this histone mark acts in cancers remains unexplored due in part to a lack of knowledge about its reader proteins. Here, we observe that cervical cancer (CC) cells undergo metabolic reprogram by which lactate accumulation and thereby boosts histone lactylation, particularly H3K14la. Utilizing a multivalent photoaffinity probe in combination with quantitative proteomics approach, we identify DPF2 as a candidate target of H3K14la. Biochemical studies as well as CUT&Tag analysis reveal that DPF2 is capable of binding to H3K14la and colocalizes with it on promoters of oncogenic genes. Notably, disrupting the DPF2–H3K14la interaction through structure-guided mutation blunts those cancer-related gene expression along with cell survival. Together, our findings reveal DPF2 as a bona fide H3K14la effector that couples histone lactylation to gene transcription and cell survival, offering insight into how histone Kla engages in transcription and tumorigenesis.