RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)-mediated protein import, nuclear export signal-mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)-importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP-importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule-kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan and induce premature ageing phenotypes and cancer in both humans and mice. A reduced BubR1 expression level is also a feature of chronological ageing, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high-level expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule-kinetochore attachment defects. Furthermore, sustained high-level expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole-chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

Several animal studies have demonstrated that mechanical thrombectomy (MT) for acute ischemic stroke (AIS) may cause vessel wall injury (VWI). However, the histological changes in human cerebral arteries following MT are difficult to determine.

PARP inhibitors (PARPi) have activity in homologous recombination (HR) repair-deficient, high-grade serous ovarian cancers (HGSOC). However, even responsive tumors develop PARPi resistance, highlighting the need to delay or prevent the appearance of PARPi resistance. Here, we showed that the ALK kinase inhibitor ceritinib synergizes with PARPis by inhibiting complex I of the mitochondrial electron transport chain, which increases production of reactive oxygen species (ROS) and subsequent induction of oxidative DNA damage that is repaired in a PARP-dependent manner. In addition, combined treatment with ceritinib and PARPi synergized in HGSOC cell lines irrespective of HR status, and a combination of ceritinib with the PARPi olaparib induced tumor regression more effectively than olaparib alone in HGSOC patient-derived xenograft (PDX) models. Notably, the ceritinib and olaparib combination was most effective in PDX models with preexisting PARPi sensitivity and was well tolerated. These findings unveil suppression of mitochondrial respiration, accumulation of ROS, and subsequent induction of DNA damage as novel effects of ceritinib. They also suggest that the ceritinib and PARPi combination warrants further investigation as a means to enhance PARPi activity in HGSOC, particularly in tumors with preexisting HR defects. SIGNIFICANCE: The kinase inhibitor ceritinib synergizes with PARPi to induce tumor regression in ovarian cancer models, suggesting that ceritinib combined with PARPi may be an effective strategy for treating ovarian cancer.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes.

We previously found that preformed complexes of BAK with antiapoptotic BCL2 proteins predict BH3 mimetic sensitivities in lymphohematopoietic cells. These complexes have not previously been examined in solid tumors or in the context of conventional anticancer drugs. Here we show the relative amount of BAK found in preformed complexes with MCL1 or BCLXL varies across ovarian cancer cell lines and patient-derived xenografts (PDXs). Cells bearing BAK/MCL1 complexes were more sensitive to paclitaxel and the MCL1 antagonist S63845. Likewise, PDX models with BAK/MCL1 complexes were more likely to respond to paclitaxel. Mechanistically, BIM induced by low paclitaxel concentrations interacted preferentially with MCL1 and displaced MCL1-bound BAK. Further studies indicated that cells with preformed BAK/MCL1 complexes were sensitive to the paclitaxel/S63845 combination, while cells without BAK/MCL1 complexes were not. Our study suggested that the assessment of BAK/MCL1 complexes might be useful for predicting response to paclitaxel alone or in combination with BH3 mimetics.

Acquired PARP inhibitor (PARPi) resistance in BRCA1- or BRCA2-mutant ovarian cancer often results from secondary mutations that restore expression of functional protein. RAD51C is a less commonly studied ovarian cancer susceptibility gene whose promoter is sometimes methylated, leading to homologous recombination (HR) deficiency and PARPi sensitivity. For this study, the PARPi-sensitive patient-derived ovarian cancer xenograft PH039, which lacks HR gene mutations but harbors RAD51C promoter methylation, was selected for PARPi resistance by cyclical niraparib treatment in vivo. PH039 acquired PARPi resistance by the third treatment cycle and grew through subsequent treatment with either niraparib or rucaparib. Transcriptional profiling throughout the course of resistance development showed widespread pathway level changes along with a marked increase in RAD51C mRNA, which reflected loss of RAD51C promoter methylation. Analysis of ovarian cancer samples from the ARIEL2 Part 1 clinical trial of rucaparib monotherapy likewise indicated an association between loss of RAD51C methylation prior to on-study biopsy and limited response. Interestingly, the PARPi resistant PH039 model remained platinum sensitive. Collectively, these results not only indicate that PARPi treatment pressure can reverse RAD51C methylation and restore RAD51C expression, but also provide a model for studying the clinical observation that PARPi and platinum sensitivity are sometimes dissociated.

The CCNE1 locus, which encodes cyclin E1, is amplified in many types of cancer cells and is activated in hepatocellular carcinomas (HCCs) from patients infected with hepatitis B virus or adeno-associated virus type 2, due to integration of the virus nearby. We investigated cell-cycle and oncogenic effects of cyclin E1 overexpression in tissues of mice.

Reduced BRCA1 expression causes homologous recombination (HR) repair defects in high-grade serous ovarian cancers (HGSOCs). Here, we demonstrate that BRCA1 is transcriptionally activated by a previously unknown function of ZC3H18. We show that ZC3H18 is a DNA-binding protein that interacts with an E2F site in the BRCA1 promoter where it facilitates recruitment of E2F4 to an adjacent E2F site to promote BRCA1 transcription. Consistent with ZC3H18 role in activating BRCA1 expression, ZC3H18 depletion induces BRCA1 promoter methylation, reduces BRCA1 expression, disrupts HR, and sensitizes cells to DNA crosslinkers and poly(ADP-ribose) polymerase inhibitors. Moreover, in patient-derived xenografts and primary HGSOC tumors, ZC3H18 and E2F4 mRNA levels are positively correlated with BRCA1 mRNA levels, further supporting ZC3H18 role in regulating BRCA1. Given that ZC3H18 lies within 16q24.2, a region with frequent copy number loss in HGSOC, these findings suggest that ZC3H18 copy number losses could contribute to HR defects in HGSOC.

During the past decade, the age-adjusted mortality rate for endometrial cancer (EC) increased 1.9% annually with TP53 mutant (TP53-mu) EC disproportionally represented in advanced disease and deaths. Therefore, we aimed to assess pivotal molecular parameters that differentiate clinical outcomes in high- and low-risk EC. Using the Cancer Genome Atlas, we analyzed EC specimens with available DNA sequences and quantitative gene-specific RNA expression data. After polymerase ɛ (POLE)-mutant specimens were excluded, differential gene-specific mutations and mRNA expressions were annotated and integrated. Consequent to TP53-mu failure to induce p21, derepression of multiple oncogenes harboring promoter p21 repressive sites was observed, including CCNA2 and FOXM1 (P < .001 compared with TP53 wild type [TP53-wt]). TP53-wt EC with high CCNA2 expression (CCNA2-H) had a targeted transcriptomic profile similar to that of TP53-mu EC, suggesting CCNA2 is a seminal determinant for both TP53-wt and TP53-mu EC. CCNA2 enhances E2F1 function, upregulating FOXM1 and CIP2A, as observed in TP53-mu and CCNA2-H TP53-wt EC (P < .001). CIP2A inhibits protein phosphatase 2A, leading to AKT inactivation of GSK3β and restricted oncoprotein degradation; PPP2R1A and FBXW7 mutations yield similar results. Upregulation of FOXM1 and failed degradation of FOXM1 is evidenced by marked upregulation of multiple homologous recombination genes (P < .001). Integrating these molecular aberrations generated a molecular biomarker panel with significant prognostic discrimination (P = 5.8×10-7); adjusting for age, histology, grade, myometrial invasion, TP53 status, and stage, only CCNA2-H/E2F1-H (P = .0003), FBXW7-mu/PPP2R1A-mu (P = .0002), and stage (P = .017) were significant. The generated prognostic molecular classification system identifies dissimilar signaling aberrations potentially amenable to targetable therapeutic options.

High stage and recurrent ovarian clear cell carcinoma (OCC) are associated with poor prognosis and resistance to chemotherapy. A distinguishing histological feature of OCC is abundant cytoplasmic stores of glucose, in the form of glycogen, that can be mobilized for cellular metabolism. Here, we report the effect on preclinical models of OCC of disrupting glycogen utilization using the glucose analogue 2-deoxy-D-glucose (2DG). At concentrations significantly lower than previously reported for other cancers, 2DG markedly improves the efficacy in vitro of carboplatin chemotherapy against chemo-sensitive TOV21G and chemo-resistant OVTOKO OCC cell lines, and this is accompanied by the depletion of glycogen. Of note, 2DG doses-of more than 10-fold lower than previously reported for other cancers-significantly improve the efficacy of carboplatin against cell line and patient-derived xenograft models in mice that mimic the chemo-responsiveness of OCC. These findings are encouraging, in that 2DG doses, which are substantially lower than previously reported to cause adverse events in cancer patients, can safely and significantly improve the efficacy of carboplatin against OCC. Our results thus justify clinical trials to evaluate whether low dose 2DG improves the efficacy of carboplatin in OCC patients.

Poly(ADP-ribose) polymerase (PARP) inhibitors have yielded encouraging responses in high-grade serous ovarian carcinomas (HGSOCs), but the optimal treatment setting remains unknown. We assessed the effect of niraparib on HGSOC patient-derived xenograft (PDX) models as well as the relationship between certain markers of homologous recombination (HR) status, including BRCA1/2 mutations and formation of RAD51 foci after DNA damage, and response of these PDXs to niraparib in vivo.

Ovarian carcinosarcoma is a rare subtype of ovarian cancer with poor clinical outcomes. The low incidence of this disease makes accrual to large clinical trials challenging. However, studies have shown that treatment responses in patient-derived xenograft (PDX) models correlate with matched-patient responses in the clinic, supporting their use for preclinical testing of standard and novel therapies. An ovarian carcinosarcoma PDX is presented herein and showed resistance to carboplatin and paclitaxel (similar to the patient) but exhibited significant sensitivity to ifosfamide and paclitaxel. The PDX demonstrated overexpression of EGFR mRNA and gene amplification by array comparative genomic hybridization (log2 ratio 0.399). EGFR phosphorylation was also detected. Angiogensis and insulin-like growth factor pathways were also implicated by overexpression of VEGFC and IRS1. In order to improve response to chemotherapy, the PDX was treated with carboplatin/paclitaxel with or without a pan-HER and VEGF inhibitor (BMS-690514) but there was no tumor growth inhibition or improved animal survival, which may be explained by a KRAS mutation. Resistance was also observed when the IGF-1R inhibitor BMS-754807 was combined with carboplatin/paclitaxel. Because poly (ADP-ribose) polymerase inhibitors have activity in ovarian cancer patients, with and without BRCA mutations, ABT-888 was also tested but found to have no activity. Pathogenic mutations were also detected in TP53 and PIK3CA. In conclusion, ifosfamide/paclitaxel was superior to carboplatin/paclitaxel in this ovarian carcinosarcoma PDX and gene overexpression or amplification alone was not sufficient to predict response to targeted therapy. Better predictive markers of response are needed.

Gene fusions play a critical role in some cancers and can serve as important clinical targets. In epithelial ovarian cancer (EOC), the contribution of fusions, especially by histological type, is unclear. We therefore screened for recurrent fusions in a histologically diverse panel of 220 EOCs using RNA sequencing. The Pipeline for RNA-Sequencing Data Analysis (PRADA) was used to identify fusions and allow for comparison with The Cancer Genome Atlas (TCGA) tumors. Associations between fusions and clinical prognosis were evaluated using Cox proportional hazards regression models. Nine recurrent fusions, defined as occurring in two or more tumors, were observed. CRHR1-KANSL1 was the most frequently identified fusion, identified in 6 tumors (2.7% of all tumors). This fusion was not associated with survival; other recurrent fusions were too rare to warrant survival analyses. One recurrent in-frame fusion, UBAP1-TGM7, was unique to clear cell (CC) EOC tumors (in 10%, or 2 of 20 CC tumors). We found some evidence that CC tumors harbor more fusions on average than any other EOC histological type, including high-grade serous (HGS) tumors. CC tumors harbored a mean of 7.4 fusions (standard deviation [sd] = 7.4, N = 20), compared to HGS EOC tumors mean of 2.0 fusions (sd = 3.3, N = 141). Few fusion genes were detected in endometrioid tumors (mean = 0.24, sd = 0.74, N = 55) or mucinous tumors (mean = 0.25, sd = 0.5, N = 4) tumors. To conclude, we identify one fusion at 10% frequency in the CC EOC subtype, but find little evidence for common (> 5% frequency) recurrent fusion genes in EOC overall, or in HGS subtype-specific EOC tumors.

MISIIR is a potential target for ovarian cancer (OC) therapy due to its tissue-specific pattern of expression. 3C23K is a novel therapeutic monoclonal anti-MISIIR antibody designed to recruit effector cells and promote cell death through ADCC (antibody dependent cell-mediated cytotoxicity). Our objective was to determine the tolerability and efficacy of 3C23K in OC patient-derived xenografts (PDX) and to identify factors affecting efficacy. Quantitative RT-PCR, immunohistochemistry (IHC), and flow cytometry were used to categorize MISIIR expression in established PDX models derived from primary OC patients. We selected two high expressing models and two low expressing models for in vivo testing. One xenograft model using an MISIIR over-expressing SKOV3ip cell line (Z3) was a positive control. The primary endpoint was change in tumor size. The secondary endpoint was final tumor mass. We observed no statistically significant differences between control and treated animals. The lack of response could be secondary to a number of variables including the lack of known biomarkers of response, the low membrane expression of MISIIR, and a limited ability of 3C23K to induce ADCC in PDX models. Further study is needed to determine the magnitude of ovarian cancer response to 3C23K and also if there is a threshold surface expression to predict response.

As patient derived xenograft (PDX) models are increasingly used for preclinical drug development, strategies to account for the nonhuman component of PDX RNA expression data are critical to its interpretation. A bioinformatics pipeline to separate donor tumor and mouse stroma transcriptome profiles was devised and tested. To examine the molecular fidelity of PDX versus donor tumors, we compared mRNA differences between paired PDX-donor tumors from nine ovarian cancer patients. 1,935 differentially expressed genes were identified between PDX and donor tumors. Over 90% (n = 1767) of these genes were down-regulated in PDX models and enriched in stroma-specific functions. Several protein kinases were also differentially expressed in PDX tumors, e.g. PDGFRA, PDGFRB and CSF1R. Upon in silico removal of these PDX-donor tumor differentially expressed genes, a stronger transcriptional resemblance between PDX-donor tumor pairs was seen (average correlation coefficient increases from 0.91 to 0.95). We devised and validated an effective bioinformatics strategy to separate mouse stroma expression from human tumor expression for PDX RNAseq. In addition, we showed most of the PDX-donor differentially expressed genes were implicated in stromal components. The molecular similarities and differences between PDX and donor tumors have implications in future therapeutic trial designs and treatment response evaluations using PDX models.

Long-term treatment outcomes for patients with high grade ovarian cancers have not changed despite innovations in therapies. There is no recommended assay for predicting patient response to second-line therapy, thus clinicians must make treatment decisions based on each individual patient. Patient-derived xenograft (PDX) tumors have been shown to predict drug sensitivity in ovarian cancer patients, but the time frame for intraperitoneal (IP) tumor generation, expansion, and drug screening is beyond that for tumor recurrence and platinum resistance to occur, thus results do not have clinical utility. We describe a drug sensitivity screening assay using a drug delivery microdevice implanted for 24 h in subcutaneous (SQ) ovarian PDX tumors to predict treatment outcomes in matched IP PDX tumors in a clinically relevant time frame. The SQ tumor response to local microdose drug exposure was found to be predictive of the growth of matched IP tumors after multi-week systemic therapy using significantly fewer animals (10 SQ vs 206 IP). Multiplexed immunofluorescence image analysis of phenotypic tumor response combined with a machine learning classifier could predict IP treatment outcomes against three second-line cytotoxic therapies with an average AUC of 0.91.

DNA polymerase epsilon (POLE) is encoded by the POLE gene, and POLE-driven tumors are characterized by high mutational rates. POLE-driven tumors are relatively common in endometrial and colorectal cancer, and their presence is increasingly recognized in ovarian cancer (OC) of endometrioid type. POLE-driven cases possess an abundance of TCT > TAT and TCG > TTG somatic mutations characterized by mutational signature 10 from the Catalog of Somatic Mutations in Cancer (COSMIC). By quantifying the contribution of COSMIC mutational signature 10 in RNA sequencing (RNA-seq) we set out to identify POLE-driven tumors in a set of unselected Mayo Clinic OC.

Resistance to platinum compounds is a major determinant of patient survival in high-grade serous ovarian cancer (HGSOC). To understand mechanisms of platinum resistance and identify potential therapeutic targets in resistant HGSOC, we generated a data resource composed of dynamic (±carboplatin) protein, post-translational modification, and RNA sequencing (RNA-seq) profiles from intra-patient cell line pairs derived from 3 HGSOC patients before and after acquiring platinum resistance. These profiles reveal extensive responses to carboplatin that differ between sensitive and resistant cells. Higher fatty acid oxidation (FAO) pathway expression is associated with platinum resistance, and both pharmacologic inhibition and CRISPR knockout of carnitine palmitoyltransferase 1A (CPT1A), which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. The results are further validated in patient-derived xenograft models, indicating that CPT1A is a candidate therapeutic target to overcome platinum resistance. All multiomic data can be queried via an intuitive gene-query user interface (https://sites.google.com/view/ptrc-cell-line).

One of the greatest barriers to curative treatment of neuroblastoma is its frequent metastatic outgrowth prior to diagnosis, especially in cases driven by amplification of the MYCN oncogene. However, only a limited number of regulatory proteins that contribute to this complex MYCN-mediated process have been elucidated. Here we show that the growth arrest-specific 7 (GAS7) gene, located at chromosome band 17p13.1, is preferentially deleted in high-risk MYCN-driven neuroblastoma. GAS7 expression was also suppressed in MYCN-amplified neuroblastoma lacking 17p deletion. GAS7 deficiency led to accelerated metastasis in both zebrafish and mammalian models of neuroblastoma with overexpression or amplification of MYCN. Analysis of expression profiles and the ultrastructure of zebrafish neuroblastoma tumors with MYCN overexpression identified that GAS7 deficiency led to (i) downregulation of genes involved in cell-cell interaction, (ii) loss of contact among tumor cells as critical determinants of accelerated metastasis, and (iii) increased levels of MYCN protein. These results provide the first genetic evidence that GAS7 depletion is a critical early step in the cascade of events culminating in neuroblastoma metastasis in the context of MYCN overexpression. SIGNIFICANCE: Heterozygous deletion or MYCN-mediated repression of GAS7 in neuroblastoma releases an important brake on tumor cell dispersion and migration to distant sites, providing a novel mechanism underlying tumor metastasis in MYCN-driven neuroblastoma.See related commentary by Menard, p. 2815.