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High-grade serous carcinoma (HGSC) is the most common and lethal ovarian cancer subtype. PARP inhibitors (PARPi) have become the mainstay of HGSC-targeted therapy, given that these tumors are driven by a high degree of genomic instability (GI) and homologous recombination (HR) defects. Nonetheless, approximately 30% of patients initially respond to treatment, ultimately relapsing with resistant disease. Thus, despite recent advances in drug development and an increased understanding of genetic alterations driving HGSC progression, mortality has not declined, highlighting the need for novel therapies. Using a small-molecule activator of protein phosphatase 2A (PP2A; SMAP-061), we investigated the mechanism by which PP2A stabilization induces apoptosis in patient-derived HGSC cells and xenograft (PDX) models alone or in combination with PARPi. We uncovered that PP2A genes essential for cellular transformation (B56α, B56γ, and PR72) and basal phosphatase activity (PP2A-A and -C) are heterozygously lost in the majority of HGSC. Moreover, loss of these PP2A genes correlates with worse overall patient survival. We show that SMAP-061–induced stabilization of PP2A inhibits the HR output by targeting RAD51, leading to chronic accumulation of DNA damage and ultimately apoptosis. Furthermore, combination of SMAP-061 and PARPi leads to enhanced apoptosis in both HR-proficient and HR-deficient HGSC cells and PDX models. Our studies identify PP2A as a novel regulator of HR and indicate PP2A modulators as a therapeutic therapy for HGSC. In summary, our findings further emphasize the potential of PP2A modulators to overcome PARPi insensitivity, given that targeting RAD51 presents benefits in overcoming PARPi resistance driven by BRCA1/2 mutation reversions.

The large majority of high grade serous ovarian cancer (HGSOC) patients will eventually become resistant to platinum-based chemotherapy, highlighting the urgent need to develop novel therapeutics targeting recurrent, platinum-resistant disease. To study this, our lab generated two patient derived platinum-resistant tumor spheres from platinum-sensitive primary patient cells to better understand the biology behind recurrent disease and identify novel therapeutic targets. Utilizing an artificial intelligence (AI) powered model, we screened a library that consisted of compounds predicted to target known or postulated resistance mechanisms and involved a novel therapeutic target. From this screen, we identified a TNIK kinase inhibitor as the only compound to effectively kill HGSOC spheres while sparing non-malignant fallopian tube cells. TNIK plays a key role in Wnt pathway activation, which our lab has previously shown to be critical in driving cancer stemness and epithelial ovarian cancer platinum resistance. Additionally, patient data shows that TNIK is amplified in ovarian cancer more than any other cancer type and is highly activated in ovarian cancer patients. We found that this compound was able to decrease overall Wnt activity in our platinum-resistant cells and could resensitize our platinum-resistant cells to cisplatin. This work demonstrates the abilities of using AI to expedite the drug discovery process as well as uncover novel therapeutic targets which may lead to future therapeutic strategies for HGSOC patients who no longer respond to standard of care platinum chemotherapy. Citation Format: Noah Puleo, Michele Cusato, Dylan Carvette, Chantelle Husdon, Analisa DiFeo. Unraveling the function of TRAF2 and NCK interacting kinase (TNIK) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1679.

While the rates of new cases and deaths for high grade serous ovarian cancer (HGSC) have declined steadily in the past decade, HGSC remains the fifth leading cause of cancer death in women. As many patients succumb to chemoresistance, there is a need to uncover novel targeted drugs. One way to do so is by drug repurposing. A computational screen, DrugPredict, led to the discovery that amiodarone, an antiarrhythmic, is a potent apoptosis-inducer and Myc-degrader in numerous patient-derived ovarian cancer cell lines. In search of a more selective, but less toxic compound, structure-activity relationship was applied to identify DL78. Due to its structural differences, DL78 is considered to be independent of amiodarone. DL78 lacks hERG activity, retains Myc regulation, and gains selective anticancer properties. This compound produces significant cell death selectively in patient-derived HGSC cells, including isogenic cisplatin-resistant lines, but does not harm non-transformed fallopian tube cells. Cancer cells treated with DL78 arrest prior to anaphase and showcase microtubule abnormalities and upregulation of mitotic regulators. The prolonged arrest leads to loss of Myc, mitotic catastrophe, and apoptosis selectively in the HGSC cells, while fallopian tube cells can cycle normally. DL78’s activity is confirmed to be dependent on Myc expression because targeted inhibition of Myc decreases its potency and prevents the DL78-induced cell cycle arrest. Finally, pharmacokinetic analysis shows that DL78 saturates the tumor following intraperitoneal injection, yet drug concentrations in the blood and liver present at normal levels. Overall, these phenotypes do not show with amiodarone treatment, suggesting that structurally different DL78 performs through a different mechanism. Further classification of this tumor-selective compound may reveal targeted ovarian cancer vulnerabilities. Citation Format: Jessica Teitel, Michele Cusato, Margaret Farah, Agharnan Gandhi, Pil Lee, Andrew White, John Takyi-Williams, Bo Wen, Andre Monterio Da Rocha, Analisa DiFeo. Novel Myc-dependent compound DL78 selectively induces mitotic catastrophe and cell death in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1570.

Background: Hepatocellular carcinoma (HCC) is the fourth leading cause of death from cancer worldwide, and for advanced HCC the prognosis is poor. Preliminary studies indicate mistletoe extracts may have anticancer activity for HCC. Methods: A prospective observational case series of advanced HCC patients that chose to take a mistletoe extract called viscum fraxini-2 (VF-2) alone for treatment. Time on treatment, imaging, and laboratory values were collected for descriptive analyses. Results: A total of 12 patients with advanced HCC enrolled onto the protocol, and 10 patients had data available for evaluation. The majority were male (10/12) with a median age of 64 (SD 11). Most patients had received sorafenib therapy (9/12) and had varying Child-Pugh classes (A-4, B-6, C-2). Treatment with VF-2 ranged from 1 to 36 weeks with a mean of 12.3 weeks (SD 12). Six patients received 8 weeks of treatment, and 3 patients received 12 or more weeks of treatment. For patients that received at least 4 weeks of treatment, the average AFP value stabilized during the first 4 weeks of treatment. Two patients experienced an AFP decrease of >30%, approximately 37 and 40% decreases at the nadir. One patient had stable disease of 9 months. Major side effects were fever, fatigue, rash, and local injection site reaction of swelling, redness, and tenderness. Conclusion: This case series of advanced HCC indicates that mistletoe extract VF-2 may have potential biological activity against HCC for selected patients. Research is needed to identify the active compound and predictive markers of response.

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, which encodes the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30% to 40% of USC cases, the clinical actionability of these mutations has not been studied. Using a high-throughput screening approach, we showed that mutations in Aα results in synthetic lethality following treatment with inhibitors of ribonucleotide reductase (RNR). In vivo, multiple models of Aα mutant uterine serous tumors were sensitive to clofarabine, an RNR inhibitor (RNRi). Aα-mutant cells displayed impaired checkpoint signaling upon RNRi treatment and subsequently accumulated more DNA damage than wild-type (WT) cells. Consistently, inhibition of PP2A activity using LB-100, a catalytic inhibitor, sensitized WT USC cells to RNRi. Analysis of The Cancer Genome Atlas data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of patients with USC harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNRi are not routinely used for uterine cancers, a retrospective analysis of patients treated with gemcitabine as a second- or later-line therapy revealed a trend for improved outcomes in patients with USC treated with RNRi gemcitabine compared with patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. Significance: A drug repurposing screen identifies synthetic lethal interactions in PP2A-deficient uterine serous carcinoma, providing potential therapeutic avenues for treating this deadly endometrial cancer.

Uterine serous carcinoma (USC) is a highly aggressive endometrial cancer subtype with limited therapeutic options and a lack of targeted therapies. While mutations to PPP2R1A, encoding the predominant protein phosphatase 2A (PP2A) scaffolding protein Aα, occur in 30-40% of cases, the clinical actionability of these mutations has not been studied. Here, we show that mutation to Aα results in synthetic lethality to treatment with inhibitors of ribonucleotide reductase (RNR), and multiple models of Aα mutant uterine serous tumors were sensitive to Clofarabine, an RNR inhibitor in vivo. Aα mutant cells displayed impaired checkpoint signaling upon RNRi treatment, and subsequently accumulated more DNA damage than wild type cells. This was PP2A dependent as complete inhibition of PP2A activity using LB-100, a catalytic site inhibitor, sensitized wild type USC cells to RNRi. Analysis of TCGA data indicated that inactivation of PP2A, through loss of PP2A subunit expression, was prevalent in USC, with 88% of USC patients harboring loss of at least one PP2A gene. In contrast, loss of PP2A subunit expression was rare in uterine endometrioid carcinomas. While RNR inhibitors are not routinely used for uterine cancers, we identified a cohort of patients with recurrent disease treated with gemcitabine at MSKCC as a second or later line therapy. In a retrospective analysis of this cohort there was a trend for improved outcomes in USC patients treated with RNRi gemcitabine compared to patients with endometrioid histology. Overall, our data provide experimental evidence to support the use of ribonucleotide reductase inhibitors for the treatment of USC. Citation Format: Caitlin M. O'Connor, Sarah E. Taylor, Kathryn M. Miller, Lauren Hurst, Terrance J. Haanen, Tahra K. Suhan, Kaitlin P. Zawacki, Fallon K. Noto, Jonida Trako, Arathi Mohan, Jaya Sangodkar, Dmitriy Zamarin, Analisa DiFeo, Goutham Narla. Synthetic lethality by targeting ribonucleotide reductase in PP2A deficient uterine serous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3341.

For decades, chemotherapy has remained the standard of care for ovarian cancer patients. Although 70% of patients initially respond to platinum-based therapy, >90% succumb to chemoresistance. There is a need to uncover targeted drugs for ovarian cancer. A quicker, more cost-effective, and lower-risk route to identify new treatments is through repurposing FDA approved drugs. Using a computational drug repositioning platform, DrugPredict, we previously uncovered that the antiarrhythmic drug amiodarone potently decreases cell viability and triggers apoptosis in numerous patient-derived ovarian cancer cell lines through the degradation of c-Myc. However, given the dose-limiting toxicity of amiodarone, we applied structure-activity relationship to identify DL78, which lacks hERG activity but retains the anti-cancer properties and ability to regulate Myc. DL78 is significantly more potent and tumor specific than amiodarone. It rapidly induces G2/M arrest which ultimately leads to loss of Myc, mitotic catastrophe, and apoptosis in several types of cancer cells. Furthermore, pharmacokinetics studies show that the compound is retained in the tumor upon intraperitoneal injection and has reasonable solubility and permeability, yielding good absorption, distinct from amiodarone. Given its structural and molecular differences, we expect that DL78 works through a different mechanism that amiodarone and could represent an effective treatment for ovarian cancer and other MYC-driven tumors. Additional studies will establish the foundation for further development of this novel compound, as well as reveal targeted ovarian cancer vulnerabilities. Citation Format: Jessica McAnulty, Michele Dziubinski, Agharnan Gandhi, Margaret Farah, Pil Lee, Andrew D. White, Analisa DiFeo. Identification of a targeted anti-mitotic agent that degrades Myc and specifically induces cancer cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4003.

High Grade Serous Carcinoma (HGSC) is the most lethal ovarian cancer subtype and accounts for approximately 60% of all ovarian tumors. Despite recent advances in drug development and increased understanding of genetic alterations that drive HGSC progression, mortality has not declined, highlighting the need for novel therapies. PARP inhibitors (PARPi) have become the mainstay of HGSC targeted therapy research given that these tumors are driven by a high degree of genomic instability resulting from the combination of fast DNA replication rates and numerous defects in the DNA-damage response (DDR) pathway. Nonetheless, only ~25% of these patients initially respond to treatment and a significant percentage eventually relapses with resistant disease. Here, we discovered that a Small Molecule Activator of Protein Phosphatase 2A (PP2A) (SMAP-061) induces apoptosis in both established and patient-derived HGSC cell lines as well as in genetically distinct Patient-Derived Xenograft (PDX) mouse models. Interestingly, we also uncovered that several genes that make-up the heterotrimer PP2A tumor suppressor protein are heterozygously lost in more than 95% of HGSC tumors, second only to p53. Mechanistically, we show that stabilization of PP2A protein by SMAP-061 inhibits the Homologous Recombination (HR) pathway via the direct inhibition of RAD51, ultimately leading to chronic accumulation of DNA damage and thus programmed cell death. Furthermore, we found that SMAP-061’s ability to inhibit HR potentiated the effects of PARP inhibition and resulted in synergistic cell death in both HR proficient and deficient models. These studies emphasize the potential of PP2A activators to expand the patient population that can benefit from PARPi therapies and possibly overcome PARPi resistance. In sum, our data highlights a new role of PP2A in regulating the DDR pathway in HGSC and supports the use of SMAPs in both HR proficient and deficient HGSC tumors. Citation Format: Rita A. Avelar, Amy Armstrong, Goutham Narla, Analisa DiFeo. Small molecule mediated stabilization of PP2A modulates the homologous recombination pathway and potentiates DNA damage-induced cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3340.

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a preclinical research model including larger sample size collection, lower rates of drug clearance, and relative ease of surgical manipulation. An immunodeficient rat would allow for larger tumor size development, prolonged dosing and drug efficacy studies, and preliminary toxicologic testing and pharmacokinetic/pharmacodynamic studies in the same model animal. Here, we created an immunodeficient rat with a functional deletion of the Recombination Activating Gene 2 (Rag2) gene, using genetically modified spermatogonial stem cells (SSC). We targeted the Rag2 gene in rat SSCs with TALENs and transplanted these Rag2-deficient SSCs into sterile recipients. Offspring were genotyped, and a founder with a 27 bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2tm1Hera (SDR) knockout rat. We demonstrated that SDR rat lacks mature B and T cells. Furthermore, the SDR rat model was permissive to growth of human glioblastoma cell line subcutaneously resulting in successful growth of tumors. In addition, a human KRAS-mutant non–small cell lung cancer cell line (H358), a patient-derived high-grade serous ovarian cancer cell line (OV81), and a patient-derived recurrent endometrial cancer cell line (OV185) were transplanted subcutaneously to test the ability of the SDR rat to accommodate human xenografts from multiple tissue types. All human cancer cell lines showed efficient tumor uptake and growth kinetics indicating that the SDR rat is a viable host for a range of xenograft studies. Mol Cancer Ther; 17(11); 2481–9. ©2018 AACR.

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks.

Wnt signaling is a major driver of stemness and chemoresistance in ovarian cancer, yet the genetic drivers that stimulate its expression remain largely unknown. Unlike other cancers, mutations in the Wnt pathway are not reported in high-grade serous ovarian cancer (HGSOC). Hence, a key challenge that must be addressed to develop effective targeted therapies is to identify nonmutational drivers of Wnt activation. Using an miRNA sensor-based approach, we have identified miR-181a as a novel driver of Wnt/β-catenin signaling. miR-181ahigh primary HGSOC cells exhibited increased Wnt/β-catenin signaling, which was associated with increased stem-cell frequency and platinum resistance. Consistent with these findings, inhibition of β-catenin decreased stem-like properties in miR-181ahigh cell populations and downregulated miR-181a. The Wnt inhibitor SFRP4 was identified as a novel target of miR-181a. Overall, our results demonstrate that miR-181a is a nonmutational activator of Wnt signaling that drives stemness and chemoresistance in HGSOC, suggesting that the miR–181a–SFRP4 axis can be evaluated as a novel biomarker for β-catenin–targeted therapy in this disease. Significance: These results demonstrate that miR-181a is an activator of Wnt signaling that drives stemness and chemoresistance in HGSOC and may be targeted therapeutically in recurrent disease.

Purpose: Acquired resistance to cisplatin is a major barrier to success in treatment of various cancers, and understanding mitotic mechanisms unique to cisplatin-resistant cancer cells can provide the basis for developing novel mitotic targeted therapies aimed at eradicating these cells. Experimental Design: Using cisplatin-resistant models derived from primary patient epithelial ovarian cancer (EOC) cells, we have explored the status of mitotic exit mechanisms in cisplatin-resistant cells. Results: We have uncovered an unexpected role of long-term cisplatin treatment in inducing mitotic exit vulnerability characterized by increased spindle checkpoint activity and functional dependency on Polo-like kinase 1 (PLK1) for mitotic exit in the presence of anaphase promoting complex/cyclosome (APC/C) dysfunction in a cisplatin-resistant state. Accordingly, PLK1 inhibition decreased the survival of cisplatin-resistant cells in vitro and in vivo and exacerbated spindle checkpoint response in these cells. APC/CCDC20 inhibition increased sensitivity to pharmacologic PLK1 inhibition, further confirming the existence of APC/C dysfunction in cisplatin-resistant cells. In addition, we uncovered that resistance to volasertib, PLK1 inhibitor, is due to maintenance of cells with low PLK1 expression. Accordingly, stable PLK1 downregulation in cisplatin-resistant cells induced tolerance to volasertib. Conclusions: We provide the first evidence of APC/C dysfunction in cisplatin-resistant state, suggesting that understanding APC/C functions in cisplatin-resistant state could provide a basis for developing novel mitotic exit–based therapies to eradicate cisplatin-resistant cancer cells. Our results also show that PLK1 downregulation could underlie emergence of resistance to PLK1-targeted therapies in cancers. Clin Cancer Res; 24(18); 4588–601. ©2018 AACR.

Large-scale chromosomal alterations, particularly chromosomal deletions in cancer genomes confer functional advantages to cancer cells via the loss of tumor suppressor genes (TSGs). However, due to the nature of these focal and arm-level deletions, essential house-keeping genes in the neighborhood of TSGs are potentially lost. We explore the emergence of metabolic adaptations and vulnerabilities that arise due to the collateral loss of essential metabolic genes. In our previous work, we showed that genomic loss in the locus containing SMAD4 and ME2 in pancreatic ductal adenocarcinomas forces these cells to rely on ME3 to compensate for the collateral loss of ME2; thereby revealing a highly selective metabolic target in these cells. Cancer cells can not only exploit such genetic redundancies but also rely on redundancies built into their complex metabolic network to compensate for the loss of metabolic function. Importantly, there is an unexplored landscape of these genomic loss events beyond well-characterized TSGs. To address these challenges, we have developed a platform to identify patient-specific metabolic vulnerabilities emerging due to distinct patterns of genomic loss events across tumors. Our platform presents opportunities for precision-based therapeutic intervention by targeting metabolic vulnerabilities in cancer patients. It uses genomic and clinical data available in cancer patient databases to obtain candidate metabolic genes that are lost to genomic deletions in an unbiased manner. To delineate metabolic redundances and tackle the complexity of genome-scale metabolic models, we employ an innovative multi-objective metabolic flux analysis approach. The utility of this platform is demonstrated via the discovery of a novel metabolic target in a cohort of ovarian cancer patients. The predicted collateral lethal target is validated in vitro using RNA interference and small-molecule inhibitors. Furthermore, we verify the metabolic mechanism of vulnerability predicted by the algorithm using deuterium tracing experiments. The target is also validated in vivo with mice containing ovarian tumors derived from cancer cells with and without the genomic deletion. Surprisingly, the collateral lethal metabolic target was also found to exist in a subset of aggressive endometrial cancers. Finally, we developed a multi-layer machine learning model to predict occurrence of the particular genomic deletion in ovarian and endometrial cancer patients with minimal molecular information to remove the need for whole-genome sequencing data. The model was trained and tested using the publicly-available molecular data from TCGA and AACR GENIE datasets. Citation Format: Abhinav Achreja, Tao Yu, Anjali Mittal, Srinadh Choppara, Noah Meurs, Olamide Animasahun, Jin Heon Jeon, Aradhana Mohan, Anusha Jayaraman, Reva Kulkarni, Justin Reinhold, Michele Cusato, Analisa Difeo, Xiongbin Lu, Deepak Nagrath. Genomic loss in cancers enable discovery of metabolic targets for precision cancer therapy via multiobjective flux analysis and machine learning [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 236.

Chemoresistance is driven by unique regulatory networks in the genome that are distinct from those necessary for cancer development. Here, we investigate the contribution of enhancer elements to cisplatin resistance in ovarian cancers. Epigenome profiling of multiple cellular models of chemoresistance identified unique sets of distal enhancers, super-enhancers (SE), and their gene targets that coordinate and maintain the transcriptional program of the platinum-resistant state in ovarian cancer. Pharmacologic inhibition of distal enhancers through small-molecule epigenetic inhibitors suppressed the expression of their target genes and restored cisplatin sensitivity in vitro and in vivo. In addition to known drivers of chemoresistance, our findings identified SOX9 as a critical SE-regulated transcription factor that plays a critical role in acquiring and maintaining the chemoresistant state in ovarian cancer. The approach and findings presented here suggest that integrative analysis of epigenome and transcriptional programs could identify targetable key drivers of chemoresistance in cancers. Significance: Integrative genome-wide epigenomic and transcriptomic analyses of platinum-sensitive and -resistant ovarian lines identify key distal regulatory regions and associated master regulator transcription factors that can be targeted by small-molecule epigenetic inhibitors.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and, very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel (n-PTX) has been added to the arsenal of first-line therapies, and the combination of gemcitabine and n-PTX has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying n-PTX resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naïve patients with PDAC. To study the mechanisms of primary drug resistance, nab-paclitaxel–resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the n-PTX-R cells. Depletion of c-MYC restored n-PTX sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small-molecule activator of protein phosphatase 2a. Implications: The strategies we have devised, including the patient-derived primary cells and the unique, drug-resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.

Somatic mutation of the protein phosphatase 2A (PP2A) Aα-subunit gene PPP2R1A is highly prevalent in high-grade endometrial carcinoma. The structural, molecular, and biological basis by which the most recurrent endometrial carcinoma–specific mutation site P179 facilitates features of endometrial carcinoma malignancy has yet to be fully determined. Here, we used a series of structural, biochemical, and biological approaches to investigate the impact of the P179R missense mutation on PP2A function. Enhanced sampling molecular dynamics simulations showed that arginine-to-proline substitution at the P179 residue changes the protein's stable conformation profile. A crystal structure of the tumor-derived PP2A mutant revealed marked changes in A-subunit conformation. Binding to the PP2A catalytic subunit was significantly impaired, disrupting holoenzyme formation and enzymatic activity. Cancer cells were dependent on PP2A disruption for sustained tumorigenic potential, and restoration of wild-type Aα in a patient-derived P179R-mutant cell line restored enzyme function and significantly attenuated tumorigenesis and metastasis in vivo. Furthermore, small molecule–mediated therapeutic reactivation of PP2A significantly inhibited tumorigenicity in vivo. These outcomes implicate PP2A functional inactivation as a critical component of high-grade endometrial carcinoma disease pathogenesis. Moreover, they highlight PP2A reactivation as a potential therapeutic strategy for patients who harbor P179R PPP2R1A mutations. Significance: This study characterizes a highly recurrent, disease-specific PP2A PPP2R1A mutation as a driver of endometrial carcinoma and a target for novel therapeutic development. See related commentary by Haines and Huang, p. 4009

Objective To evaluate the utility of amiodarone and its derivative dronedarone as novel drug repositioning candidates in EOC and to determine the potential pathways targeted by these drugs. Methods Drug-predict bioinformatics platform was used to assess the utility of amiodarone as a novel drug-repurposing candidate in EOC. EOC cells were treated with amiodarone and dronedarone. Cell death was assessed by Annexin V staining. Cell viability and cell survival were assessed by MTT and clonogenics assays respectively. c-MYC and mTOR/Akt axis were evaluated as potential targets. Effect on autophagy was determined by autophagy flux flow cytometry. Results "DrugPredict" bioinformatics platform ranked Class III antiarrhythmic drug amiodarone within the top 3.9% of potential EOC drug repositioning candidates which was comparable to carboplatin ranking in the top 3.7%. Amiodarone and dronedarone were the only Class III antiarrhythmic drugs that decreased the cellular survival of both cisplatin-sensitive and cisplatin-resistant primary EOC cells. Interestingly, both drugs induced degradation of c-MYC protein and decreased the expression of known transcriptional targets of c-MYC. Furthermore, stable overexpression of non-degradable c-MYC partially rescued the effects of amiodarone and dronedarone induced cell death. Dronedarone induced higher autophagy flux in EOC cells as compared to amiodarone with decreased phospho-AKT and phospho-4EBP1 protein expression, suggesting autophagy induction due to inhibition of AKT/mTOR axis with these drugs. Lastly, both drugs also inhibited the survival of EOC tumor-initiating cells (TICs). Conclusions We provide the first evidence of class III antiarrhythmic agents as novel c-MYC targeting drugs and autophagy inducers in EOC. Since c-MYC is amplified in >40% ovarian tumors, our results provide the basis for repositioning amiodarone and dronedarone as novel c-MYC targeting drugs in EOC with potential extension to other cancers.

Genomic instability predisposes cells to malignant transformation, however the molecular mechanisms that allow for the propagation of cells with a high-degree of genomic instability remains unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells (FTSECs, the precursor cell type for the majority of high-grade serous ovarian cancers) through the inhibition of RB1. Increased miR-181a expression simultaneously drives cell protective inhibition of the stimulator-of-interferon-genes (STING) in order to maintain a microenvironment conducive to the propagation of cells with a high-degree of genomic instability. We found that miR-181a inhibition of RB1 leads to profound nuclear defects, genomic instability, and nuclear rupture resulting in a persistence of genomic material in the cytoplasm. Normally, this persistence of genomic material in the cytoplasm induces a cell-intrinsic interferon response through STING to drive cell death. However, miR-181a directly downregulates STING and prevents interferon induction and cell death in the FTSECs. The most common mechanism by which oncogenic miRNAs promote tumorigenesis is through the direct inhibition of tumor suppressor genes. However, our studies highlight a new mechanism of oncomiR transformation through the combination of tumor suppressor gene and intrinsic interferon signaling inhibition that initiates and propagates tumor cell survival. Importantly, we found that miR-181a induction in ovarian patient tumors is tightly associated with decreased IFNγ response and downregulation of lymphocyte infiltration and leukocyte fraction. Our findings are the first to identify a miRNA, miR-181a, that can downregulate STING expression and suppress activation of cell-intrinsic innate immune signaling in precursor cells undergoing oncogenic transformation to create a survival advantage. Our studies support the notion that the induction of STING-mediated signaling in developing cancer cells could lead directly to cancer cell death, and that these effects can be inhibited by miR-181a. Given the recent interest in the development of STING agonists as a strategy to harness the immune system to treat cancer, this study introduces miR-181a as a novel patient selective biomarker as well as potential therapeutic target for HGSOC treatment. Citation Format: Matthew Knarr, Rita Avelar, Sreeja Sekhar, Lily Kwiatkowski, Michele Dziubinski, Jessica McAnulty, Stephanie Skala, Stefanie Avril, Ronny Drapkin, Analisa DiFeo. miR-181a initiates and perpetuates ovarian cancer transformation through inhibition of the tumor suppressors RB1 and stimulator of interferon genes (STING) [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-076.

// Chantelle D. Hudson 1 , Alyssa Savadelis 1 , Anil Belur Nagaraj 2 , Peronne Joseph 2 , Stefanie Avril 3 , Analisa DiFeo 2, * , Norbert Avril 1, * 1 Department of Radiology, Case Center for Imaging Research, Case Western Reserve University, Cleveland, OH 44106, USA 2 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44106, USA 3 Department of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA * Shared senior authors Correspondence to: Norbert Avril, email: Norbert.Avril@Case.edu Keywords: ovarian cancer, cisplatin resistance, glutamine metabolism, glutaminase inhibitors Received: February 13, 2016 Accepted: April 10, 2016 Published: May 12, 2016 ABSTRACT Ovarian cancer is characterized by an increase in cellular energy metabolism, which is predominantly satisfied by glucose and glutamine. Targeting metabolic pathways is an attractive approach to enhance the therapeutic effectiveness and to potentially overcome drug resistance in ovarian cancer. In platinum-sensitive ovarian cancer cell lines the metabolism of both, glucose and glutamine was initially up-regulated in response to platinum treatment. In contrast, platinum-resistant cells revealed a significant dependency on the presence of glutamine, with an upregulated expression of glutamine transporter ASCT2 and glutaminase. This resulted in a higher oxygen consumption rate compared to platinum-sensitive cell lines reflecting the increased dependency of glutamine utilization through the tricarboxylic acid cycle. The important role of glutamine metabolism was confirmed by stable overexpression of glutaminase, which conferred platinum resistance. Conversely, shRNA knockdown of glutaminase in platinum resistant cells resulted in re-sensitization to platinum treatment. Importantly, combining the glutaminase inhibitor BPTES with platinum synergistically inhibited platinum sensitive and resistant ovarian cancers in vitro . Apoptotic induction was significantly increased using platinum together with BPTES compared to either treatment alone. Our findings suggest that targeting glutamine metabolism together with platinum based chemotherapy offers a potential treatment strategy particularly in drug resistant ovarian cancer.

// Andrew Wiechert 1, 2, * , Caner Saygin 1, * , Praveena S. Thiagarajan 1 , Vinay S. Rao 1 , James S. Hale 1 , Nikhil Gupta 3 , Masahiro Hitomi 1, 3, 4 , Anil Belur Nagaraj 5 , Analisa DiFeo 5 , Justin D. Lathia 1, 3, 4, 5 , Ofer Reizes 1, 3, 4, 5 1 Department of Cellular and Molecular Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA 2 Division of Gynecological Oncology, Women’s Health Institute, Cleveland Clinic, Cleveland, OH 44195, USA 3 Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195 4 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland, OH 44195, USA 5 Department of Case Comprehensive Cancer Center, Cleveland, OH 44106, USA * These authors contributed equally to this work Correspondence to: Ofer Reizes, e-mail: reizeso@ccf.org Justin D. Lathia, e-mail: lathiaj@ccf.org Keywords: cancer stem cell, ovarian cancer, cisplatin, NANOG Received: December 01, 2015 Accepted: March 31, 2016 Published: April 20, 2016 ABSTRACT The mainstay of treatment for ovarian cancer is platinum-based cytotoxic chemotherapy. However, therapeutic resistance and recurrence is a common eventuality for nearly all ovarian cancer patients, resulting in poor median survival. Recurrence is postulated to be driven by a population of self-renewing, therapeutically resistant cancer stem cells (CSCs). A current limitation in CSC studies is the inability to interrogate their dynamic changes in real time. Here we utilized a GFP reporter driven by the NANOG-promoter to enrich and track ovarian CSCs. Using this approach, we identified a population of cells with CSC properties including enhanced expression of stem cell transcription factors, self-renewal, and tumor initiation. We also observed elevations in CSC properties in cisplatin-resistant ovarian cancer cells as compared to cisplatin-naive ovarian cancer cells. CD49f, a marker for CSCs in other solid tumors, enriched CSCs in cisplatin-resistant and -naive cells. NANOG-GFP enriched CSCs (GFP+ cells) were more resistant to cisplatin as compared to GFP-negative cells. Moreover, upon cisplatin treatment, the GFP signal intensity and NANOG expression increased in GFP-negative cells, indicating that cisplatin was able to induce the CSC state. Taken together, we describe a reporter-based strategy that allows for determination of the CSC state in real time and can be used to detect the induction of the CSC state upon cisplatin treatment. As cisplatin may provide an inductive stress for the stem cell state, future efforts should focus on combining cytotoxic chemotherapy with a CSC targeted therapy for greater clinical utility.

c-Myc is part of the Myc family proteins associated with cellular proliferation, differentiation, and apoptosis. Dysregulated c-Myc expression is associated with tumorigenesis, and cancers with c-Myc overexpression are often associated with poor prognosis, partly due to difficulty in targeting c-Myc. Mistletoe lectin (ML), derived from the European mistletoe plant, has shown variable efficacy in cancer patients. One case series following 12 patients with advanced hepatocellular carcinoma (HCC) showed 2 patients with &gt30% reduction in the tumor marker alpha-fetoprotein and 1 patient who had stable disease for 9 months. These cases indicate promising results. ML consists of a heteroprotein complex of an A- and B-chain connected by a disulfide bond. The A-chain was previously described to inhibit translation by 28s rRNA cleavage, while the B-chain binds cell surface glycoproteins to trigger internalization of the lectin. We found that purified mistletoe extract, consisting primarily of ML, downregulates c-Myc protein and induces apoptosis in in vitro and in vivo models of human-derived cancer, and that the efficacy of ML treatment is strongly correlated with MYC gene expression and c-Myc protein abundance in in vitro human models of HCC, ovarian cancer, small cell lung cancer, and lymphoma. MTS assay data demonstrated that exogenous MYC expression can sensitize low-MYC cell lines to ML treatment, suggesting that anti-cancer effects of ML are dependent on its ability to downregulate c-Myc. While ML may be able to downregulate many proteins via general translational inhibition, we found that of 296 proteins profiled by RPPA, c-Myc was one of 11 proteins decreased by ML with at least a 2-fold change (p&lt0.01). We also found that ML promotes c-Myc ubiquitylation, suggesting that ML regulates c-Myc protein stability, rather than synthesis. In summary, here we describe a novel mechanism of action for ML in downregulating c-Myc and promoting apoptotic cell death in various models of MYC-driven cancer, and provide evidence for MYC gene expression as a predictive marker for response. This research supports the development of ML-based therapies that may provide a c-Myc-targeting treatment in multiple cancer types. Citation Format: Gabrielle Kennelley, Eric Yuan, Mohammad Shatat, Betsy Gauthier, Peiying Yang, Analisa Difeo, Richard Lee. Mistletoe lectin induces apoptosis in MYC-driven cancers through post-translational downregulation of c-MYC protein [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1831.

Genomic instability predisposes cells to malignant transformation; however, the molecular mechanisms that allow for the propagation of cells with a high degree of genomic instability remain unclear. Here we report that miR-181a is able to transform fallopian tube secretory epithelial cells—the precursor cell type for the majority of high-grade serous ovarian cancers—through the inhibition of RB1 and simultaneously drives a cell-protective inhibition of the stimulator-of-interferon-genes (STING) in order to maintain a microenvironment conducive to the propagation of cells with a high-degree of genomic instability. We found that miR-181a inhibition of RB1 leads to profound nuclear defects, genomic instability, and nuclear rupture resulting in a persistence of genomic material in the cytoplasm. While normally, this persistence of genomic material in the cytoplasm induces interferon response through STING to drive cell death, miR-181a directly downregulates STING and prevents apoptosis. The most common mechanism by which oncogenic miRNAs promote tumorigenesis is through the direct inhibition of tumor suppressor genes; however, our studies highlight a new mechanism of oncomiR transformation through the combination of tumor suppressor gene inhibition and abrogation of immune surveillance that initiates and propagates tumor cell survival. Importantly, we found that miR-181a induction in human ovarian tumors is tightly associated with decreased IFNg response and downregulation of lymphocyte infiltration and leukocyte fraction. To date, DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation; thus, our findings are the first to identify a genetic factor, miR-181a, that can downregulate STING expression and impair signaling in cancer cells, creating a survival advantage. Our studies support the notion that the induction of STING-mediated signaling in cancer cells could lead directly to cancer cell death; however, these effects are abrogated by miR-181a. Given the recent interest in the development of STING agonists as a strategy to harness the immune system to treat cancer, this study introduces a novel patient-selective biomarker as well as potent therapeutic target for development of the most effective combination treatments. Citation Format: Matthew Knarr, Lily J. Kwiatkowski, Michele Dziubinski, Jessica McAnulty, Stephanie Skala, Stefanie Avril, Ronny Drapkin, Analisa DiFeo. miR-181a initiates and perpetuates oncogenic transformation through the regulation of innate immune signaling [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B02.

Ovarian cancer (OvC) is the deadliest of all female gynecologic malignancies, ranking as the fifth cause of cancer-related deaths among women. High-grade serous epithelial ovarian cancer (HGSOC) accounts for almost 90% of all forms of OvC, remaining as the most lethal subtype of ovarian malignancies. HGSOC is usually diagnosed at a later stage at which the disease has already progressed into metastatic, having a 5-year survival rate of only 17%. Treatment of HGSOC relies on aggressive approaches including surgery and platinum-based chemotherapies, with the majority of cases requiring combination of both. The molecular mechanism driving ovarian tumorigenesis is not fully understood, and its investigation is essential to provide better and novel therapeutic options for the treatment and cure of this disease. Targeted therapies have been vastly explored in the cancer research field due to their promise in reducing side effects by specifically targeting oncogenic pathways predominantly deregulated in human malignancies. DNA damage and repair (DDR) pathway aberrations and genomic instability are two main hallmarks of OvC. Extensive efforts in targeting this pathway have been devoted to introduce new drugs in the clinic that can target the DDR and homologous recombination (HR) pathways as therapeutic means for treatment of HGSOC. The use of PARP inhibitors (PARPi) to achieve synthetic lethality in OvC has been shown to have great promise in efficiently reducing tumor burden by specifically targeting tumor cells for cell death. However, PARPi’s antitumoral efficacy alone is limited by the status of the BRCA1/2 genes, narrowing down the patient population that can benefit from such therapies. Eventually, patients treated with PARPi alone relapse and develop resistant OvC, for which treatment options available are extremely limited. Our preliminary data show that first-in-class small-molecule activators of PP2A (SMAPs) in combination with PARPi can effectively reduce tumor burden in PDX models, promoting a synergistic effect in platinum-sensitive and -resistant OvC tumors, despite the BRCA gene status. Protein Phosphatase 2A (PP2A) and Protein Phosphatase 1 (PP1) account for 90% of all serine/threonine phosphatase activity in the cell and act as two main tumor suppressors. These phosphatases tightly regulate signaling pathways involved with cell cycle, growth, migration, metabolism, survival and many oncogenic pathways by counteracting kinases. HGSOC tumors express inactivated PP2A due to PPP2RA1 heterozygous loss, although lacking mutations, making it an ideal target for reactivation in these cancers. Moreover, we have shown that reactivation of PP2A via SMAPs leads to potent inhibition of several key DDR proteins. We have found that SMAPs treatment in BRCA1/2 WT tumors confers a “BRCAness” phenotype, which sensitizes tumors to DNA repair inhibitors such as PARPi. In sum, SMAPs can allow us to expand the patient population that can benefit from PARPi therapies and possibly overcome drug resistance. Citation Format: Rita A. Avelar, Amy Armstrong, Goutham Narla, Analisa DiFeo. Targeting Protein Phosphatase 2A in Combination with PARP inhibitors for the treatment of high-grade serous epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A34.

Effective practice of precision medicine in oncology relies on identifying and targeting unique characteristics of individual cancers. Coupling functional analysis with genomic screening will further enhance the selection of efficacious cancer therapies. Cancer is a heterogeneous disease that is defined by distinct capabilities, one of which is the evasion of apoptosis, controlled by the antiapoptotic protein members, Bcl-2, Bcl-xL, Mcl-1, and Bfl-1. These antiapoptotic proteins have become validated therapeutic targets, as manifested by the FDA-approved venetoclax, a selective Bcl-2 inhibitor, and several other molecules in clinical trials that target Mcl-1 and Bcl-xL. One of the translational challenges lies in predicting functional antiapoptotic dependence in a patient’s cancer and discriminating responders from nonresponders to Bcl-2 family targeted therapy. We have successfully characterized an updated array of Bcl-2 family antagonists, consisting of peptides and selective small-molecule BH3-mimetics via in vitro binding assays and functional analysis using model cell lymphoma cell lines. With this selective set of chemical tools, we employed the BH3 profiling assay across a wide spectrum of hematologic and solid tumor cell lines in order to functionally dissect survival dependence. To validate the results of BH3 profiling, we performed treatments with BH3-mimetics and analyzed their ability to induce apoptosis, which further demonstrated how cancers can be differentiated and targeted based on antiapoptotic dependence. The obtained data suggest that hematologic cancer cell lines mainly rely on Mcl-1 and Bcl-2 for survival and commonly undergo apoptosis in response to single-agent inhibitors of these proteins. Solid tumor cell lines are more dependent upon Bcl-xL and Mcl-1 and certain cell lines display increased involvement of Bfl-1. These cell lines undergo apoptosis when exposed to a combination of Bcl-xL/Mcl-1 inhibitors. Single agent-responsive solid tumor cell lines are less common but can be identified by BH3 profiling. This approach was successfully applied to primary patient samples to validate clinical utility. Further applications of dynamic BH3 profiling were explored to demonstrate how various FDA-approved therapies can alter antiapoptotic dependencies and aid in the design of rational combination therapies with BH3-mimetics. This work lays a translational foundation in the field of precision medicine by incorporating Bcl-2 protein family targeting into potential personalized cancer treatments. Citation Format: Karson J. Kump, Matthew Lieberman, Rita A. Avelar, Charles Foucar, Malathi Kandarpa, Antonio Di Cristofano, Russell J. Ryan, Sami N. Malek, Ryan A. Wilcox, Dale L. Bixby, Tycel J. Phillips, Moshe Talpaz, Analisa DiFeo, Zaneta Nikolovska-Coleska. Precision medicine strategy to elucidate and target Bcl-2 prosurvival proteins in a wide spectrum of cancers [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 10.

// Anil Belur Nagaraj 1 , Peronne Joseph 1 , Olga Kovalenko 1 , Sareena Singh 2 , Amy Armstrong 2 , Raymond Redline 3 , Kimberly Resnick 2 , Kristine Zanotti 2 , Steven Waggoner 2 and Analisa DiFeo 1 1 Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH, USA 2 Department of Gynecology, Division of Gynecological Oncology, University Hospital Case Medical Center, Cleveland, OH, USA 3 Department of Pathology, University Hospital Case Medical Center, Cleveland, OH, USA Correspondence to: Analisa DiFeo, email: // Keywords : patient derived xenograft, platinum resistance, tumor spheres, cancer initiating cells, β-catenin Received : April 23, 2015 Accepted : June 01, 2015 Published : June 29, 2015 Abstract Resistance to platinum-based chemotherapy is the major barrier to treating epithelial ovarian cancer. To improve patient outcomes, it is critical to identify the underlying mechanisms that promote platinum resistance. Emerging evidence supports the concept that platinum-based therapies are able to eliminate the bulk of differentiated cancer cells, but are unable to eliminate cancer initiating cells (CIC). To date, the relevant pathways that regulate ovarian CICs remain elusive. Several correlative studies have shown that Wnt/β-catenin pathway activation is associated with poor outcomes in patients with high-grade serous ovarian cancer (HGSOC). However, the functional relevance of these findings remain to be delineated. We have uncovered that Wnt/β-catenin pathway activation is a critical driver of HGSOC chemotherapy resistance, and targeted inhibition of this pathway, which eliminates CICs, represents a novel and effective treatment for chemoresistant HGSOC. Here we show that Wnt/β-catenin signaling is activated in ovarian CICs, and targeted inhibition of β-catenin potently sensitized cells to cisplatin and decreased CIC tumor sphere formation. Furthermore, the Wnt/β-catenin specific inhibitor iCG-001 potently sensitized cells to cisplatin and decreased stem-cell frequency in platinum resistant cells. Taken together, our data is the first report providing evidence that the Wnt/β-catenin signaling pathway maintains stem-like properties and drug resistance of primary HGSOC PDX derived platinum resistant models, and therapeutic targeting of this pathway with iCG-001/PRI-724, which has been shown to be well tolerated in Phase I trials, may be an effective treatment option.

Primary prostate cancer is generally treatable by androgen deprivation therapy, however, later recurrences of castrate-resistant prostate cancer (CRPC) that are more difficult to treat nearly always occur due to aberrant reactivation of the androgen receptor (AR). In this study, we report that CRPC cells are particularly sensitive to the growth-inhibitory effects of reengineered tricyclic sulfonamides, a class of molecules that activate the protein phosphatase PP2A, which inhibits multiple oncogenic signaling pathways. Treatment of CRPC cells with small-molecule activators of PP2A (SMAP) in vitro decreased cellular viability and clonogenicity and induced apoptosis. SMAP treatment also induced an array of significant changes in the phosphoproteome, including most notably dephosphorylation of full-length and truncated isoforms of the AR and downregulation of its regulatory kinases in a dose-dependent and time-dependent manner. In murine xenograft models of human CRPC, the potent compound SMAP-2 exhibited efficacy comparable with enzalutamide in inhibiting tumor formation. Overall, our results provide a preclinical proof of concept for the efficacy of SMAP in AR degradation and CRPC treatment. Significance: A novel class of small-molecule activators of the tumor suppressor PP2A, a serine/threonine phosphatase that inhibits many oncogenic signaling pathways, is shown to deregulate the phosphoproteome and to destabilize the androgen receptor in advanced prostate cancer. Cancer Res; 78(8); 2065–80. ©2018 AACR.

Epithelial ovarian cancer (EOC) is one of the deadliest gynecological cancers currently diagnosed in women with approximately 20,000 new cases and 15,000 deaths per year. Ovarian cancer can be stratified into distinct subtypes of which high-grade serous ovarian cancer (HGSOC) comprises 90% of all cases and has the highest mortality rate. Reducing the high mortality rate associated with HGSOC currently has two primary barriers: 1) most HGSOCs are diagnosed at a post-metastatic stage and 2) up to 70% of patients recur within 5 years. Both of these limitations are due to the fact that the drivers of tumor initiation are poorly understood. In order to change the status quo of the last 30 years, a new approach that focuses on understanding the origins of HGSOC is required. Our lab has recently discovered that miR-181a promotes key hallmarks of HGSOC progression in fallopian tube secretory epithelial cells (FTSECs) which are precursor cells of HGSOC. We show that enhanced expression of miR-181a in FTSECs results in increased proliferation, survival, and anchorage independent growth in vitro. Furthermore, FTSECs with miR-181a overexpression are able to form tumors in vivo whereas FTSECs that overexpress a control scramble miRNA cannot. Using cell cycle analysis, dynamic live-cell imaging, and SNP array analysis, we also found that miR-181a overexpression increases aneuploidy and genomic instability in FTSECs. We show that miR-181a promotes defects in nuclear structure, ruptures in the nuclear envelope, as well as mitotic and cytokinetic defects affecting proper chromosome segregation. A combination of global mRNA expression profiling and target prediction software identified the tumor suppressor retinoblastoma (RB1) as a target for miR-181a mediated FTSEC tumorigenesis. Using an RB1 3’UTR luciferase assay, along with stable expression of an shRNA against RB1 in FTSECs, we are able to show that miR-181a directly targets RB1 and that knockdown of RB1 phenocopies miR-181a mediated tumorigenesis. Taken together, our data shed light on a novel mechanism that promotes genomic instability and tumorigenesis in the early phases of HGSOC development. Our results suggest miR-181a as a promising early detection biomarker as well as a prognostic tool to guide patient treatment. Citation Format: Matthew Knarr, Lily Kwiatkowski, Anil Nagaraj, Ronny Drapkin, Analisa DiFeo. miR-181a is a key driver of genomic instability and ovarian cancer tumorigenesis through the regulation of RB1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3557.

Neoadjuvant chemotherapy (NACT) has been recognized as a reliable therapeutic strategy in patients with unresectable advanced epithelial ovarian cancer (EOC). The molecular events leading to platinum (Pt) response in NACT settings have hitherto not been explored. In the present work, longitudinal changes of miRNA expression profile were investigated to identify miRNA families with prognostic role in high-grade serous EOC patients who received the NACT regimen.One hundred sixty-four matched tumor biopsies taken at initial laparoscopic evaluation and at interval-debulking surgery (IDS) after four courses of Pt-based therapy were selected from 82 stage IIIC-IV high-grade serous-EOC patients that were judged unsuitable for complete primary debulking and subjected the NACT protocol. miRNA profiling by microarray, real-time PCR and immuno-histochemical staining for Smad2 phosphorylation (P-Smad2) were used for data analysis.Analysis revealed that 369 miRNAs were differentially expressed in matched biopsies (referred to as DEMs). DEMs were not scattered across the genome, but clustered into families: miR-199, let-7, miR-30, miR-181 and miR-29. Multivariate analysis showed that miR-199a-3p, miR-199a-5p, miR-181a-5p and let-7g-5p associated with overall and progression-free survival (P0.05); miR-199a-3p, miR-199a-5p and miR-181a-5p associated with residual tumor volume and Pt-free interval (P0.05). Immuno-histochemical staining confirmed an enrichment of P-Smad2, a marker of transforming growth factor-β activation, in tumors from patients with shorter PFS and OS, and with high levels of expression of miR-181a-5p (P0.05). Kaplan-Meier curves plotting concomitant expression of P-Smad2 and miR-181a-5p show significant differences in PFS and OS compared with those depicting the expression of each biomarker alone (P0.001).This study describes several miRNA families with a prognostic role in the NACT setting. It also confirms that concomitant analysis of P-Smad2 and miR-181a-5p in surgical samples may be capable of identifying those ovarian cancer patients with poor outcome and little chance of response to Pt-based NACT.

Background Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. Patients and methods We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. Results and conclusions Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, ‘Activins/Inhibins’ and ‘Hedgehog’ signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Tumor recurrence is a major barrier towards improving patient outcome in EOC. Tumor-initiating cells (TICs) survive platinum-based therapies and contribute to tumor recurrence, at which stage the tumor is often difficult to eradicate, and thus TICs form the root cause of poor patient survival in EOC. Unfortunately, no TIC-targeting drugs to date are being evaluated in EOC patient trials. Isolating majority of TIC clones present in EOC tumors for functional analysis remains elusive to date and hence, understanding the complexity of TIC functioning remains a hurdle to be overcome in TIC therapeutics. TIC regulators can differ between tumors across patients and also in tumors within a patient. Tumor heterogeneity and TIC plasticity are the major challenges towards deciphering TIC functioning in EOC. Studying EOC cell populations with TIC properties is often limited to approaches driven by enrichment based on expression of stem-cell markers, which do not faithfully reflect TIC functioning in the context of heterogeneity and plasticity. Hence, pathway-based enrichment of TICs provide a reliable approach to functionally enrich for TIC subpopulations in EOC. miRNAs are small RNA molecules that regulate post-transcriptional gene silencing by binding mostly to the 3’UTR of their potential targets and targeting them for degradation through miRNA-induced silencing complexes (miRISCs). Isolation and characterization of TIC subpopulations in EOC based on miRNA activity can offer a reliable approach to functionally enrich for TICs in EOC. We have developed a novel miRNA-sensor based functional platform driven by miR-181a 3’UTR activity with mCHERRY fluorescence as the readout. miR-181a expression was upregulated in primary recurrent EOC tumors that were clinically resistant to platinum chemotherapy, suggesting a potential role for this miRNA in regulating TIC properties in EOC. Accordingly, miR-181a high subpopulation isolated from both cisplatin-sensitive and cisplatin-resistant/recurrent primary high-grade serous (HGSOC) ovarian tumor cells using miR-181a sensor were enriched in TIC properties in vitro and in vivo. Cisplatin treatment of primary EOC cells enriched miR-181a high subpopulation and reprogrammed miR-181a low non-TICs into miR-181a high TICs. Mechanistically, Wnt signaling was upregulated in miR-181a high ovarian tumor cells, showing that miRNA sensor platform can enrich TICs based on stem-cell pathway activity. Taken together, we provide the first evidence of TIC enrichment and analysis in EOC based on miRNA 3’UTR activity that provides with a valuable tool to broaden the understanding of TIC regulatory mechanisms in EOC in “real time,” with potential extension to other cancers by overcoming the barriers of tumor heterogeneity and TIC plasticity. miRNA sensor platform will provide a TIC pharmacologic screening tool to enable the discovery of novel miRNA-inhibiting TIC-targeting drugs in EOC. Citation Format: Anil Belur Nagaraj, Peronne Joseph, Matthew Knarr, Olga Kovalenko, Arshia Surti, Analisa DiFeo. miRNA 3’UTR activity driven enrichment of ovarian tumor-initiating cells (TICs) to overcome the barriers of heterogeneity and TIC plasticity. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B66.

Nat. Med. 24, 176–185 (2018); published online 15 January 2018; corrected after print 7 February 2018 In the version of this article initially published, two of the authors are incorrectly identified as John Stamatoyannopolus and Henri Versteeg. The authors' names are John A Stamatoyannopoulos and Henri H Versteeg.

Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Previously, we have demonstrated that increased expression of KLF6-SV1 is associated with decreased survival in lung adenocarcinoma patient samples and that targeted reduction of KLF6-SV1 using siRNA induced apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Here, we demonstrate that chemoresistant lung cancer cells express increased levels of KLF6-SV1. Furthermore, targeted reduction of KLF6-SV1 using RNA interference restores chemotherapy sensitivity to lung cancer cells both in culture and in vivo through induction of apoptosis. Conversely, overexpression of KLF6-SV1 resulted in a marked reduction in chemotherapy sensitivity in a tumor xenograft model. Combined, these findings highlight a functional role for the KLF6-SV1 splice variant in the regulation of chemotherapy response in lung cancer and could provide novel insight into lung cancer therapy.

Epidemiologic evidence suggests that a diet rich in fruits and vegetables is associated with a reduced risk of prostate cancer (PCa) development. Although several dietary compounds have been tested in preclinical PCa prevention models, no agents have been identified that either prevent the progression of premalignant lesions or treat advanced disease. Momordica charantia, known as bitter melon in English, is a plant that grows in tropical areas worldwide and is both eaten as a vegetable and used for medicinal purposes. We have isolated a protein, designated as MCP30, from bitter melon seeds. The purified fraction was verified by SDS-PAGE and mass spectrometry to contain only 2 highly related single chain Type I ribosome-inactivating proteins (RIPs), alpha-momorcharin and beta-momorcharin. MCP30 induces apoptosis in PIN and PCa cell lines in vitro and suppresses PC-3 growth in vivo with no effect on normal prostate cells. Mechanistically, MCP30 inhibits histone deacetylase-1 (HDAC-1) activity and promotes histone-3 and -4 protein acetylation. Treatment with MCP30 induces PTEN expression in a prostatic intraepithelial neoplasia (PIN) and PCa cell lines resulting in inhibition of Akt phosphorylation. In addition, MCP30 inhibits Wnt signaling activity through reduction of nuclear accumulation of beta-catenin and decreased levels of c-Myc and Cyclin-D1. Our data indicate that MCP30 selectively induces PIN and PCa apoptosis and inhibits HDAC-1 activity. These results suggest that Type I RIPs derived from plants are HDAC inhibitors that can be utilized in the prevention and treatment of prostate cancer.

Defects in apoptosis are not only a hallmark of cancer initiation and progression but can also underlie the development of chemoresistance. How the tightly regulated cascade of protein-protein interactions between members of three competing protein families regulating the apoptotic cascade is subverted in tumor cells is incompletely understood. Here, we show that KLF6-SV1, whose overexpression is associated with poor survival in several different cancers and is an alternatively spliced isoform of the Krüppel-like tumor suppressor KLF6, is a critical prosurvival/antiapoptotic protein. KLF6-SV1 binds the proapoptotic BH3-only protein NOXA, which results in their mutual HDM2-dependent degradation. In turn, this increases the intracellular concentration of the prosurvival binding partner of NOXA, Mcl-1, and effectively blocks apoptosis. In an ovarian cancer model, systemically delivered small interfering RNA against KLF6-SV1 induces spontaneous apoptosis of tumor cells, decreases tumor burden, and restores cisplatin sensitivity in vivo. Moreover, i.p. delivery of siKLF6-SV1 RNA halts ovarian tumor progression and improves median and overall survival (progression-free for >15 months; P < 0.0002) in mice in a dose-dependent manner. Thus, KLF6-SV1 represents a novel regulator of protein interactions in the apoptotic cascade and a therapeutically targetable control point. [Cancer Res 2009;69(11):4733–41]

Gastric cancer is the second most common cancer and a leading cause of cancer-related death worldwide. The Kruppel-like factor 6 (KLF6) tumour suppressor gene had been previously shown to be inactivated in a number of human cancers through loss of heterozygosity (LOH), somatic mutation, decreased expression and increased alternative splicing into a dominant negative oncogenic splice variant, KLF6-SV1. In the present study, 37 gastric cancer samples were analysed for the presence of loss of heterozygosity (LOH) of the KLF6 locus and somatic mutation. In total, 18 of 34 (53%) of the gastric cancer samples analysed demonstrated KLF6 locus specific loss. Four missense mutations, such as T179I, R198G, R71Q and S180L, were detected. Interestingly, two of these mutations R71Q and S180L have been identified independently by several groups in various malignancies including prostate, colorectal and gastric cancers. In addition, decreased wild-type KLF6 (wtKLF6) expression was associated with loss of the KLF6 locus and was present in 48% of primary gastric tumour samples analysed. Functional studies confirmed that wtKLF6 suppressed proliferation of gastric cancer cells via transcriptional regulation of the cyclin-dependent kinase inhibitor p21 and the oncogene c-myc. Functional characterisation of the common tumour-derived mutants demonstrated that the mutant proteins fail to suppress proliferation and function as dominant negative regulators of wtKLF6 function. Furthermore, stable overexpression of the R71Q and S180L tumour-derived mutants in the gastric cancer cell line, Hs746T, resulted in an increased tumourigenicity in vivo. Combined, these findings suggest an important role for the KLF6 tumour suppressor gene in gastric cancer development and progression and identify several highly cancer-relevant signalling pathways regulated by the KLF6 tumour suppressor gene.

The Krüppel-like zinc finger transcription factor (KLF6) gene encodes a family of proteins generated through alternative splicing involved in the regulation of cancer development and progression. Alternative splicing of the KLF6 gene results in the production of at least four alternatively spliced isoforms, two of which are extensively discussed in this review. The full length form of the KLF6 gene is a tumor suppressor gene that is frequently inactivated by loss of heterozygozity (LOH), somatic mutation, and/or decreased expression in human cancer. While the exact mechanisms underlying KLF6's tumor suppressor roles are not completely known, a number of highly relevant, overlapping pathways have been described: transactivation of p21 in a p53-independent manner, reduction of cyclin D1/cdk4 complexes via interaction with cyclin D1, inhibition of c-Jun proto-oncoprotein activities, decreased VEGF expression, and induction of apoptosis. Kruppel-like factor 6 splice variant 1 (KLF6-SV1) is an oncogenic splice variant of the KLF6 tumor suppressor gene that is specifically overexpressed in a number of human cancers. Increased KLF6-SV1 expression is associated with poor prognosis in prostate, lung, and ovarian cancer. Furthermore, KLF6-SV1 has been shown to be biologically active, antagonizing the tumor suppressor function of KLF6 and promoting tumor growth and dissemination in both ovarian and prostate cancer models. In addition, a common germline polymorphism in the KLF6 gene associated with increased prostate cancer risk in a large multi-institutional study of 3411 men results in increased expression of KLF6-SV1. Furthermore, recent studies have demonstrated that targeted reduction of KLF6-SV1 results in the induction of spontaneous apoptosis in cell culture, synergizes with chemotherapeutic agents like cisplatin, and results in significant tumor regression in vivo. Combined, these data make the KLF6 gene family a compelling therapeutic target for both the treatment of localized as well as metastatic cancer.

Purpose: We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC).Experimental Design: We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided.Results: LOH was present in 59% of samples in a cell type–specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased ∼5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation.Conclusion: Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.

Prostate cancer is a leading and increasingly prevalent cause of cancer death in men. Whereas family history of disease is one of the strongest prostate cancer risk factors and suggests a hereditary component, the predisposing genetic factors remain unknown. We first showed that KLF6 is a tumor suppressor somatically inactivated in prostate cancer and since then, its functional loss has been further established in prostate cancer cell lines and other human cancers. Wild-type KLF6, but not patient-derived mutants, suppresses cell growth through p53-independent transactivation of p21. Here we show that a germline KLF6 single nucleotide polymorphism, confirmed in a tri-institutional study of 3,411 men, is significantly associated with an increased relative risk of prostate cancer in men, regardless of family history of disease. This prostate cancer–associated allele generates a novel functional SRp40 DNA binding site and increases transcription of three alternatively spliced KLF6 isoforms. The KLF6 variant proteins KLF6-SV1 and KLF6-SV2 are mislocalized to the cytoplasm, antagonize wtKLF6 function, leading to decreased p21 expression and increased cell growth, and are up-regulated in tumor versus normal prostatic tissue. Thus, these results are the first to identify a novel mechanism of self-encoded tumor suppressor gene inactivation and link a relatively common single nucleotide polymorphism to both regulation of alternative splicing and an increased risk in a major human cancer.

Computation-based drug repositioning approaches that automatically search vast amounts of genomic-chemical-phenotypic data for tens of thousands of drugs and diseases can greatly speed up the traditional drug-discovery process. To date, systematic and comprehensive computation-based approaches to identify and validate drug repositioning candidates for High-grade serous ovarian cancer (HGSOC) have not been undertaken. Using a novel computational drug-repositioning platform (Drug-Predict) with HGSOC gene expression dataset from The Cancer Genome Atlas (TCGA) as the input, we have uncovered that Non-steroidal anti-inflammatory drugs (NSAIDs) could be potential candidates for Drug repositioning in HGSOC. Given that numerous epidemiological studies have shown that regular intake of NSAIDs in women is associated with decreased incidence of ovarian cancer, we assessed whether NSAIDs could have chemo-adjuvant applications in HGSOC and have identified Indomethacin as a novel chemo-adjuvant in HGSOC. Indomethacin decreased survival of primary HGSOC tumor cells and interestingly, cisplatin resistant ovarian tumor cells (derived from primary HGSOC PDX models) exhibited significantly higher cell death upon Indomethacin treatment suggesting that Indomethacin could exert chemo-adjuvant effects in HGSOC. Accordingly, Indomethacin treatment induced chemo-sensitivity in cisplatin resistant HGSOC tumor cells and combo treatment with Indomethacin and cisplatin exerted synergistic cell death as compared to individual drugs alone. Furthermore, Indomethacin decreased stem-like properties and induced chemo-sensitivity in ALDHhigh cisplatin-resistant tumor-initiating cells (TICs) thus suggesting that the chemo-adjuvant effect of the drug is mediated by decrease in TIC properties in HGSOC cells. Mechanistically, we found that Indomethacin inhibits Wnt/β-catenin signaling by degrading β-catenin and β-catenin modulation inversely affected Indomethacin functioning in HGSOC. Our study is the first report describing functional effects of an NSAID in patient-derived HGSOC models and demonstrates that combining novel computational predictions with experimental validation has potential in identifying viable drug candidates and moving them into patient trials efficiently and cost-effectively. Since NSAIDs are in routine clinical use in gynecological settings and have acceptable safety profile, this discovery provides with a potentially rapid and cost-effective translational opportunity for testing NSAIDs as chemo-adjuvants in patient trials in HGSOC. Citation Format: Anil Belur Nagaraj, QuanQiu Wang, Olga Kovalenko, Peronne Joseph, Yang Chen, Rong Xu and Analisa DiFeo. PHARMACOLOGIC ANALYSIS OF HIGH–GRADE SEROUS OVARIAN CANCER TCGA DATASET IDENTIFIES A NOVEL CHEMO–ADJUVANT ROLE FOR NON–STEROIDAL ANTI–INFLAMMATORY DRUGS (NSAIDS) [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-081.

e17054 Background: Racial disparities in outcomes exist between African-American (AA) and Caucasian (C) patients (pts) with high grade epithelial ovarian cancer (EOC). Not all disparity is explained by socio-economic and access related issues. Many argue that differences in tumor biology also play a role. Germline BRCA mutations confer a protective effect in these pts. Immunohistochemistry (IHC) for BRCA 1 is being developed to detect somatic BRCA1 mutations. Parp-inhibitors have been approved for use in pts with germline and/or somatic BRCA mutations. We describe the first comparison of the prognostic implications of BRCA1 somatic mutation status in tumor samples taken from AA and C EOC pts who underwent primary surgery followed by systemic chemotherapy. Methods: IHC staining of whole tumor sections from AA and C pts was performed using a commercially available monoclonal antibody against BRCA1 (Calbiochem). Staining was scored as: loss, retention, or equivocal in the presence of a positive control. Retention was described as wild-type and loss or equivocal as mutation. Standard statistical analyses were used to correlate race, BRCA1 status, and survival. Results: Tumor from 33 pts (16AA and 17C) was stained and scored. Most pts had Stage III (66.7%) disease and high grade serous subtype (82%). Stage, histology, and debulking status did not differ between groups. Overall, loss of BRCA 1 occurred 16/33 (48.5%) tumors. BRCA1 was mutated more frequently in tumors from AA pts (11/16) compared to C pts (5/17) (p=.024). BRCA 1 mutation conferred a protective effect, with a trend towards improved progression free survival: 9.5 vs 15.8 mos in AA (p=0.44) and 16.5 vs. 21 mos in C pts (p=0.97). No statistically significant difference in OS was seen. Conclusions: We demonstrate that IHC for BRCA 1 mutation is feasible in a standard hospital pathology laboratory. Race based differences in somatic BRCA 1 mutation exist; mutational differences are associated with a trend in improved PFS. A larger cohort is needed to elucidate the true effect of this status on outcome. Conceivably, knowledge of BRCA 1 tumor status could be used to improve outcomes in AA pts who may be able to achieve survival numbers comparable to C pts.

Endometrial cancer is the most commonly diagnosed malignancy of the female genital tract in the United States. The role of adjuvant radiation for the treatment of patients with early stage disease is still heavily debated, as no overall survival benefit has been demonstrated in large prospective trials. There is, however, evidence to suggest a decrease in recurrence risk with administration of adjuvant radiotherapy, especially in patients with high-risk features. In this review, we describe the relevant literature to date, define how we determine which patients are considered high or intermediate risk, and describe the findings related to the addition of chemotherapy to adjuvant brachytherapy.

Patients with resected stage II-III cutaneous melanomas remain at high risk for metastasis and death. Biomarker development has been limited by the challenge of isolating high-quality RNA for transcriptome-wide profiling from formalin-fixed and paraffin-embedded (FFPE) primary tumor specimens. Using NanoString technology, RNA from 40 stage II-III FFPE primary melanomas was analyzed and a 53-immune-gene panel predictive of non-progression (area under the curve (AUC)=0.920) was defined. The signature predicted disease-specific survival (DSS P

Background Ovarian cancer is the most lethal gynecologic disease in the United States, with more women dying from this cancer than all gynecological cancers combined. Ovarian cancer has been termed the “silent killer” because some patients do not show clear symptoms at an early stage. Currently, there is a lack of approved and effective early diagnostic tools for ovarian cancer. There is also an apparent severe knowledge gap of ovarian cancer in general and of its indicative symptoms among both public and many health professionals. These factors have significantly contributed to the late stage diagnosis of most ovarian cancer patients (63% are diagnosed at Stage III or above), where the 5-year survival rate is less than 30%. The paucity of knowledge concerning ovarian cancer in the United States is unknown. Methods The present investigation examined current public awareness and knowledge about ovarian cancer. The study implemented design strategies to develop an unbiased survey with quality control measures, including the modern application of multiple statistical analyses. The survey assessed a reasonable proxy of the US population by crowdsourcing participants through the online task marketplace Amazon Mechanical Turk, at a highly condensed rate of cost and time compared to traditional recruitment methods. Conclusion Knowledge of ovarian cancer was compared to that of breast cancer using repeated measures, bias control and other quality control measures in the survey design. Analyses included multinomial logistic regression and categorical data analysis procedures such as correspondence analysis, among other statistics. We confirmed the relatively poor public knowledge of ovarian cancer among the US population. The simple, yet novel design should set an example for designing surveys to obtain quality data via Amazon Mechanical Turk with the associated analyses.

Metastasis is the major cause of cancer mortality. A more thorough understanding of the mechanisms driving this complex multistep process will aid in the identification and characterization of therapeutically targetable genetic drivers of disease progression. We demonstrate that KLF6-SV1, an oncogenic splice variant of the KLF6 tumor suppressor gene, is associated with increased metastatic potential and poor survival in a cohort of 671 lymph node–negative breast cancer patients. KLF6-SV1 overexpression in mammary epithelial cell lines resulted in an epithelial-to-mesenchymal–like transition and drove aggressive multiorgan metastatic disease in multiple in vivo models. Additionally, KLF6-SV1 loss-of-function studies demonstrated reversion to an epithelial and less invasive phenotype. Combined, these findings implicate KLF6-SV1 as a key driver of breast cancer metastasis that distinguishes between indolent and lethal early-stage disease and provides a potential therapeutic target for invasive breast cancer.

PP2A is a phosphatase that is functionally dysregulated and inactivated in over 50% of all breast cancers independent of histological type. While much attention has focused on oncogenic kinases as targets for cancer treatment, therapeutic targeting of phosphatases, the key negative regulators of these same signaling pathways, has remained largely unexplored. Starting with the observation that tricyclic neuroleptic drugs exert anticancer effects, our laboratory reverse engineered these drugs to generate a novel series of compounds that retain the anti-proliferative effects and favorable pharmacokinetic properties but are devoid of the undesirable central nervous system pharmacology of the parent drugs. It was subsequently demonstrated that these first generation novel derivatives exert anticancer effects in cell culture and xenograft models of cancer (without overt toxicity) by modulation of critical oncogenic signaling pathways. Recently, it was determined that the molecular basis of the actions of these novel derivatives is the activation of PP2A. Comprehensive profiling for activity in a panel of 240 cancer cell lines has shown that these first-in-class small molecule activators of PP2A (SMAPs) have significant anti-proliferative activity in breast cancer cell lines, and studies have been extended to disease relevant in vivo models (patient derived xenograft, GEMM, and traditional xenograft models). PP2A's ability to negatively regulate a diverse set of oncogenic drivers in breast cancer means SMAPs are able to exert their effects through multiple biological mechanisms, swiftly disrupting cellular energetics and metabolism, inducing apoptosis and inhibiting proliferation. This will be exhibited through a variety of biological assays evaluating cell growth, apoptosis and changes in cellular metabolism. Target engagement assessed via western blotting demonstrates inhibition of PP2A targets critical for mediating SMAP activity, notably c-MYC and AKT. As a result, this research demonstrates SMAPs have potent activity in a wide variety of breast cancer contexts where response to current therapies is modest and limited by the development of treatment resistance. Thus, SMAPs may be a viable treatment option for high-risk populations of patients who do not respond to the current standard of care. This research strives to demonstrate that therapeutic reactivation of PP2A may represent a novel approach for breast cancer treatment and that these molecules may favorably impact the lives of women suffering from breast cancer. Citation Format: Caroline C. Farrington, Xiaoyan Wang, Mahnaz Janghorban, Juan Liang, Analisa Difeo, Rosalie Sears, Goutham Narla. Pharmacological reactivation of the tumor suppressor protein phosphatase 2A as a novel approach for the treatment of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3857.