Germline mutations in SPRTN cause Ruijs-Aalfs syndrome (RJALS), a disorder characterized by genome instability, progeria and early onset hepatocellular carcinoma. Spartan, the protein encoded by SPRTN, is a nuclear metalloprotease that is involved in the repair of DNA-protein crosslinks (DPCs). Although Sprtn hypomorphic mice recapitulate key progeroid phenotypes of RJALS, whether this model expressing low amounts of Spartan is prone to DPC repair defects and spontaneous tumors is unknown. Here, we showed that the livers of Sprtn hypomorphic mice accumulate DPCs containing Topoisomerase 1 covalently linked to DNA. Furthermore, these mice exhibited DNA damage, aneuploidy and spontaneous tumorigenesis in the liver. Collectively, these findings provide evidence that partial loss of Spartan impairs DPC repair and tumor suppression.

How mitochondrial metabolism is altered by oncogenic tyrosine kinases to promote tumor growth is incompletely understood. Here, we show that oncogenic HER2 tyrosine kinase signaling induces phosphorylation of mitochondrial creatine kinase 1 (MtCK1) on tyrosine 153 (Y153) in an ABL-dependent manner in breast cancer cells. Y153 phosphorylation, which is commonly upregulated in HER2+ breast cancers, stabilizes MtCK1 to increase the phosphocreatine energy shuttle and promote proliferation. Inhibition of the phosphocreatine energy shuttle by MtCK1 knockdown or with the creatine analog cyclocreatine decreases proliferation of trastuzumab-sensitive and -resistant HER2+ cell lines in culture and in xenografts. Finally, we show that cyclocreatine in combination with the HER2 kinase inhibitor lapatinib reduces the growth of a trastuzumab-resistant HER2+ patient-derived xenograft. These findings suggest that activation of the phosphocreatine energy shuttle by MtCK1 Y153 phosphorylation creates a druggable metabolic vulnerability in cancer.

Both genetic and epigenetic factors influence the development and progression of epithelial ovarian cancer (EOC). However, there is an incomplete understanding of the interrelationship between these factors and the extent to which they interact to impact disease risk. In the present study, we aimed to gain insight into this relationship by identifying DNA methylation marks that are candidate mediators of ovarian cancer genetic risk.

Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLXL•BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.

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.

Caspase-activated DNase (CAD) is a major apoptotic nuclease, responsible for DNA fragmentation and chromatin condensation during apoptosis. CAD is normally activated in apoptosis as a result of caspase cleavage of its inhibitory chaperone ICAD. Other aspects of CAD regulation are poorly understood. In particular, it has been unclear whether direct CAD activation in non-apoptotic living cells can trigger cell death. Taking advantage of the auxin-inducible degron (AID) system, we have developed a suicide system with which ICAD is rapidly degraded in living cells in response to the plant hormone auxin. Our studies demonstrate that rapid ICAD depletion is sufficient to activate CAD and induce cell death in DT40 and yeast cells. In the vertebrate cells, ectopic CAD activation triggered caspase activation and subsequent hallmarks of caspase-dependent apoptotic changes, including phosphatidylserine exposure and nuclear fragmentation. These observations not only suggest that CAD activation drives apoptosis through a positive feedback loop, but also identify a unique suicide system that can be used for controlling gene-modified organisms.

Poly(ADP-ribose) polymerase (PARP) inhibitors have shown substantial activity in homologous recombination- (HR-) deficient ovarian cancer and are undergoing testing in other HR-deficient tumors. For reasons that are incompletely understood, not all patients with HR-deficient cancers respond to these agents. Preclinical studies have demonstrated that changes in alternative DNA repair pathways affect PARP inhibitor (PARPi) sensitivity in ovarian cancer models. This has not previously been assessed in the clinical setting.

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.

Although ovarian cancer is the most lethal of gynecologic malignancies, wide variation in outcome following conventional therapy continues to exist. The presence of tumor-infiltrating regulatory T cells (Tregs) has a role in outcome of this disease, and a growing body of data supports the existence of inherited prognostic factors. However, the role of inherited variants in genes encoding Treg-related immune molecules has not been fully explored. We analyzed expression quantitative trait loci (eQTL) and sequence-based tagging single nucleotide polymorphisms (tagSNPs) for 54 genes associated with Tregs in 3,662 invasive ovarian cancer cases. With adjustment for known prognostic factors, suggestive results were observed among rarer histological subtypes; poorer survival was associated with minor alleles at SNPs in RGS1 (clear cell, rs10921202, p=2.7×10(-5)), LRRC32 and TNFRSF18/TNFRSF4 (mucinous, rs3781699, p=4.5×10(-4), and rs3753348, p=9.0×10(-4), respectively), and CD80 (endometrioid, rs13071247, p=8.0×10(-4)). Fo0r the latter, correlative data support a CD80 rs13071247 genotype association with CD80 tumor RNA expression (p=0.006). An additional eQTL SNP in CD80 was associated with shorter survival (rs7804190, p=8.1×10(-4)) among all cases combined. As the products of these genes are known to affect induction, trafficking, or immunosuppressive function of Tregs, these results suggest the need for follow-up phenotypic studies.

Genome-wide association studies (GWAS) have identified four susceptibility loci for epithelial ovarian cancer (EOC), with another two suggestive loci reaching near genome-wide significance. We pooled data from a GWAS conducted in North America with another GWAS from the UK. We selected the top 24,551 SNPs for inclusion on the iCOGS custom genotyping array. We performed follow-up genotyping in 18,174 individuals with EOC (cases) and 26,134 controls from 43 studies from the Ovarian Cancer Association Consortium. We validated the two loci at 3q25 and 17q21 that were previously found to have associations close to genome-wide significance and identified three loci newly associated with risk: two loci associated with all EOC subtypes at 8q21 (rs11782652, P = 5.5 × 10(-9)) and 10p12 (rs1243180, P = 1.8 × 10(-8)) and another locus specific to the serous subtype at 17q12 (rs757210, P = 8.1 × 10(-10)). An integrated molecular analysis of genes and regulatory regions at these loci provided evidence for functional mechanisms underlying susceptibility and implicated CHMP4C in the pathogenesis of ovarian cancer.

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.

Despite low incidence, ovarian cancer is the fifth leading cause of cancer deaths and it has the highest mortality rate of all gynecologic malignancies among US women. The mortality rate would be reduced with an early detection marker. The folate receptor alpha (FRalpha) is one logical choice for a biomarker because of its prevalent overexpression in ovarian cancer and its exclusive expression in only a few normal tissues. In prior work, it was observed that patients with ovarian cancer had elevated serum levels of a protein that bound to a FRalpha-specific monoclonal antibody relative to healthy individuals. However, it was not shown that the protein detected was intact functional FRalpha. In the current study, the goal was to determine whether ovarian cancer patients (n = 30) had elevated serum levels of a fully functional intact FRalpha compared to matched healthy controls (n = 30).

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.

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state "addicted" to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-XL/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-XL-selective inhibitors such as S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.

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.

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).

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.

No abstract available

Small molecule direct BAK activators can potentially be used for the development of anti-cancer drugs or as tools to study BAK activation. The thrombopoietin receptor agonist eltrombopag (Eltro) inhibits BAX activation and BAX-mediated apoptosis. Here we report that, in contrast to its function as a BAX inhibitor, Eltro directly binds BAK but induces its activation in vitro. Moreover, Eltro induces or sensitizes BAK-dependent cell death in mouse embryonic fibroblasts (MEFs) and Jurkat cells. Chemical shift perturbation analysis by NMR indicates that Eltro binds to the BAK α4/α6/α7 groove to initiate BAK activation. Further molecular docking by HADDOCK suggests that several BAK residues, including R156, F157, and H164, play an important role in the interaction with Eltro. The introduction of an R156E mutation in the BAK α4/α6/α7 groove not only decreases Eltro binding and Eltro-induced BAK activation in vitro but also diminishes Eltro-induced apoptosis. Thus, our data suggest that Eltro directly induces BAK activation and BAK-dependent apoptosis, providing a starting point for the future development of more potent and selective direct BAK activators.