Interest in preclinical drug development for ovarian cancer has stimulated development of patient-derived xenograft (PDX) or tumorgraft models. However, the unintended formation of human lymphoma in severe combined immunodeficiency (SCID) mice from Epstein-Barr virus (EBV)-infected human lymphocytes can be problematic. In this study, we have characterized ovarian cancer PDXs which developed human lymphomas and explore methods to suppress lymphoproliferative growth. Fresh human ovarian tumors from 568 patients were transplanted intraperitoneally in SCID mice. A subset of PDX models demonstrated atypical patterns of dissemination with mediastinal masses, hepatosplenomegaly, and CD45-positive lymphoblastic atypia without ovarian tumor engraftment. Expression of human CD20 but not CD3 supported a B-cell lineage, and EBV genomes were detected in all lymphoproliferative tumors. Immunophenotyping confirmed monoclonal gene rearrangements consistent with B-cell lymphoma, and global gene expression patterns correlated well with other human lymphomas. The ability of rituximab, an anti-CD20 antibody, to suppress human lymphoproliferation from a patient's ovarian tumor in SCID mice and prevent growth of an established lymphoma led to a practice change with a goal to reduce the incidence of lymphomas. A single dose of rituximab during the primary tumor heterotransplantation process reduced the incidence of CD45-positive cells in subsequent PDX lines from 86.3% (n = 117 without rituximab) to 5.6% (n = 160 with rituximab), and the lymphoma rate declined from 11.1% to 1.88%. Taken together, investigators utilizing PDX models for research should routinely monitor for lymphoproliferative tumors and consider implementing methods to suppress their growth.

Although 70-80% of newly diagnosed ovarian cancer patients respond to first-line therapy, almost all relapse and five-year survival remains below 50%. One strategy to increase five-year survival is prolonging time to relapse by improving first-line therapy response. However, no biomarker today can accurately predict individual response to therapy. In this study, we present analytical and prospective clinical validation of a new test that utilizes primary patient tissue in 3D cell culture to make patient-specific response predictions prior to initiation of treatment in the clinic. Test results were generated within seven days of tissue receipt from newly diagnosed ovarian cancer patients obtained at standard surgical debulking or laparoscopic biopsy. Patients were followed for clinical response to chemotherapy. In a study population of 44, the 32 test-predicted Responders had a clinical response rate of 100% across both adjuvant and neoadjuvant treated populations with an overall prediction accuracy of 89% (39 of 44, p < 0.0001). The test also functioned as a prognostic readout with test-predicted Responders having a significantly increased progression-free survival compared to test-predicted Non-Responders, p = 0.01. This correlative accuracy establishes the test's potential to benefit ovarian cancer patients through accurate prediction of patient-specific response before treatment.

The fluoropyrimidines 5-fluorouracil (5-FU) and FdUrd (5-fluorodeoxyuridine; floxuridine) are the backbone of chemotherapy regimens for colon cancer and other tumors. Despite their widespread use, it remains unclear how these agents kill tumor cells. Here, we have analyzed the checkpoint and DNA repair pathways that affect colon tumor responses to 5-FU and FdUrd. These studies demonstrate that both FdUrd and 5-FU activate the ATR and ATM checkpoint signaling pathways, indicating that they cause genotoxic damage. Notably, however, depletion of ATM or ATR does not sensitize colon cancer cells to 5-FU, whereas these checkpoint pathways promote the survival of cells treated with FdUrd, suggesting that FdUrd exerts cytotoxicity by disrupting DNA replication and/or inducing DNA damage, whereas 5-FU does not. We also found that disabling the base excision (BER) repair pathway by depleting XRCC1 or APE1 sensitized colon cancer cells to FdUrd but not 5-FU. Consistent with a role for the BER pathway, we show that small molecule poly(ADP-ribose) polymerase 1/2 (PARP) inhibitors, AZD2281 and ABT-888, remarkably sensitized both mismatch repair (MMR)-proficient and -deficient colon cancer cell lines to FdUrd but not to 5-FU. Taken together, these studies demonstrate that the roles of genotoxin-induced checkpoint signaling and DNA repair differ significantly for these agents and also suggest a novel approach to colon cancer therapy in which FdUrd is combined with a small molecule PARP inhibitor.

Endometriosis is a highly prevalent, chronic, heterogeneous, fibro-inflammatory disease that remains recalcitrant to conventional therapy. We previously showed that loss of KLF11, a transcription factor implicated in uterine disease, results in progression of endometriosis. Despite extensive homology, co-expression, and human disease association, loss of the paralog Klf10 causes a unique inflammatory, cystic endometriosis phenotype in contrast to fibrotic progression seen with loss of Klf11. We identify here for the first time a novel role for KLF10 in endometriosis. In an animal endometriosis model, unlike wild-type controls, Klf10(-/-) animals developed cystic lesions with massive immune infiltrate and minimal peri-lesional fibrosis. The Klf10(-/-) disease progression phenotype also contrasted with prolific fibrosis and minimal immune cell infiltration seen in Klf11(-/-) animals. We further found that lesion genotype rather than that of the host determined each unique disease progression phenotype. Mechanistically, KLF10 regulated CD40/CD154-mediated immune pathways. Both inflammatory as well as fibrotic phenotypes are the commonest clinical manifestations in chronic fibro-inflammatory diseases such as endometriosis. The complementary, paralogous Klf10 and Klf11 models therefore offer novel insights into the mechanisms of inflammation and fibrosis in a disease-relevant context. Our data suggests that divergence in underlying gene dysregulation critically determines disease-phenotype predominance rather than the conventional paradigm of inflammation being precedent to fibrotic scarring. Heterogeneity in clinical progression and treatment response are thus likely from disparate gene regulation profiles. Characterization of disease phenotype-associated gene dysregulation offers novel approaches for developing targeted, individualized therapy for recurrent and recalcitrant chronic disease.

We describe a phase II clinical trial of the combination of ribociclib and letrozole for treatment of relapsed oestrogen receptor (ER)-positive ovarian cancer (OC) and endometrial cancer (EC). The primary endpoint was the proportion of patients alive, progression-free survival (PFS), and still on treatment at 12 weeks (PFS12), with 45% or greater considered positive.

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.

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.

Replication stress impedes DNA polymerase progression causing activation of the ataxia telangiectasia and Rad3-related signaling pathway, which promotes the intra-S phase checkpoint activity through phosphorylation of checkpoint kinase 1 (Chk1). Chk1 suppresses replication origin firing, in part, by disrupting the interaction between the preinitiation complex components Treslin and TopBP1, an interaction that is mediated by TopBP1 BRCT domain-binding to two cyclin-dependent kinase (CDK) phosphorylation sites, T968 and S1000, in Treslin. Two nonexclusive models for how Chk1 regulates the Treslin-TopBP1 interaction have been proposed in the literature: in one model, these proteins dissociate due to a Chk1-induced decrease in CDK activity that reduces phosphorylation of the Treslin sites that bind TopBP1 and in the second model, Chk1 directly phosphorylates Treslin, resulting in dissociation of TopBP1. However, these models have not been formally examined. We show here that Treslin T968 phosphorylation was decreased in a Chk1-dependent manner, while Treslin S1000 phosphorylation was unchanged, demonstrating that T968 and S1000 are differentially regulated. However, CDK2-mediated phosphorylation alone did not fully account for Chk1 regulation of the Treslin-TopBP1 interaction. We also identified additional Chk1 phosphorylation sites on Treslin that contributed to disruption of the Treslin-TopBP1 interaction, including S1114. Finally, we showed that both of the proposed mechanisms regulate origin firing in cancer cell line models undergoing replication stress, with the relative roles of each mechanism varying among cell lines. This study demonstrates that Chk1 regulates Treslin through multiple mechanisms to promote efficient dissociation of Treslin and TopBP1 and furthers our understanding of Treslin regulation during the intra-S phase checkpoint.

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.

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.

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).

The Fanconi anemia (FA) network is important for the repair of interstrand DNA cross-links. A key event in FA pathway activation is the monoubiquitylation of the FA complementation group I (FANCI)-FANCD2 (ID) complex by FA complementation group L (FANCL), an E3 ubiquitin ligase. In this study, we show that RAD18, another DNA damage-activated E3 ubiquitin ligase, also participates in ID complex activation by ubiquitylating proliferating cell nuclear antigen (PCNA) on Lys164, an event required for the recruitment of FANCL to chromatin. We also found that monoubiquitylated PCNA stimulates FANCL-catalyzed FANCD2 and FANCI monoubiquitylation. Collectively, these experiments identify RAD18-mediated PCNA monoubiquitination as a central hub for the mobilization of the FA pathway by promoting FANCL-mediated FANCD2 monoubiquitylation.

Inhibition of pregnancy-associated plasma protein-A (PAPP-A), an upstream activator of the insulin-like growth factor (IGF) pathway, is known to augment sensitivity to platinum-based chemotherapy. This study further tests the efficacy of PAPP-A inhibition with a monoclonal antibody inhibitor (mAb-PA) in ovarian cancer (OC) platinum-resistant patient-derived xenograft (PDX) models.