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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.

The serine/threonine Protein Phosphatase 2A (PP2A) functions as a tumor suppressor by negatively regulating multiple oncogenic signaling pathways. The canonical PP2A holoenzyme is comprised of a scaffolding subunit (PP2A Aα/β), which serves as the platform for binding of both the catalytic C subunit and one regulatory B subunit. Somatic heterozygous missense mutations in PPP2R1A, the gene encoding the PP2A Aα scaffolding subunit, have been identified across multiple cancer types, but the effects of the most commonly mutated residue, Arg-183, on PP2A function have yet to be fully elucidated. In this study, we used a series of cellular and in vivo models and discovered that the most frequent Aα R183W mutation formed alternative holoenzymes by binding of different PP2A regulatory subunits compared to wild type Aα, suggesting a rededication of PP2A functions. Unlike wild type Aα, which suppressed tumorigenesis, the R183W mutant failed to suppress tumor growth in vivo through activation of the MAPK pathway in RAS-mutant transformed cells. Furthermore, cells expressing R183W were less sensitive to MEK inhibitors. Taken together, our results demonstrate that the R183W mutation in PP2A Aα scaffold abrogates the tumor suppressive actions of PP2A, thereby potentiating oncogenic signaling and reducing drug sensitivity of RAS-mutant cells.

Endometrial cancer (EC) is the most common gynecologic malignancy and is the result of disruption of the balance between estrogen-stimulated growth and progesterone-induced growth modulation. Metformin has been shown to inhibit EC proliferation; however, its role in early-stage EC and its effects on steroid hormone receptors have not been adequately explored. Our aim was to examine the effects of metformin on cellular proliferation in patient-derived, low-grade EC cell lines and to determine whether it directly modulates steroid hormone receptor expression. Two novel EC cell lines were produced (EM2 and 3) from endometrial tumor tissue obtained from women undergoing surgery. Cellular proliferation was determined by the 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide assay, and in both cell lines, metformin decreased cell proliferation in a dose-dependent (10-200 µmol/L) manner and induced apoptosis as measured by cleaved PARP. Furthermore, metformin abrogated the effects of E2 on cell proliferation. Using quantitative real-time polymerase chain reaction and Western immunoblotting, metformin significantly decreased estrogen receptor (ER) α messenger RNA abundance but did not consistently affect the expression of progesterone receptor. Estrogen receptor α protein levels significantly decreased across all metformin doses tested, which resulted in a significant decrease in the expression of the ER targets genes Keratin-19 and Wnt-1 inducible signaling pathway 2. In addition, metformin increased phosphorylation of AMPK in a dose-dependent manner (10-200 µmol/L) indicating an effect on mammalian target of rapamycin (mTOR) signaling. Our data suggest that metformin therapy represents a potential fertility-sparing option for women with early-stage EC, given its capacity to inhibit EC cell proliferation, ERα expression, and the mTOR cell proliferation pathway.

Summary Tumor-initiating cells (TICs) contribute to drug resistance and tumor recurrence in cancers, thus experimental approaches to dissect the complexity of TICs are required to design successful TIC therapeutic strategies. Here, we show that miRNA-3′ UTR sensor vectors can be used as a pathway-based method to identify, enrich, and analyze TICs from primary solid tumor patient samples. We have found that an miR-181ahigh subpopulation of cells sorted from primary ovarian tumor cells exhibited TIC properties in vivo, were enriched in response to continuous cisplatin treatment, and showed activation of numerous major stem cell regulatory pathways. This miRNA-sensor-based platform enabled high-throughput drug screening leading to identification of BET inhibitors as transcriptional inhibitors of miR-181a. Taken together, we provide a valuable miRNA-sensor-based approach to broaden the understanding of complex TIC regulatory mechanisms in cancers and to identify miRNA-targeting drugs., Highlights • miRNA sensors provide a novel approach to identify TICs in primary human tumors • miR-181a is a driver of tumor initiation in ovarian cancer • miRNA sensors allow discovery of miRNA transcriptional regulators and targeting drugs • BET inhibitors are novel miR-181a-targeting drugs, In this article, DiFeo and colleagues have utilized a miRNA 3′ UTR biosensor to study tumor-initiating cells in ovarian cancer and develop a high-throughput platform to uncover miRNA-targeting drugs. They have identified miR-181a as a potent driver of tumor initiation, which can be regulated by BET inhibitors, thus introducing a previously unknown biomarker for these drugs.

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.

While we appreciate the importance of independent validation of scientific discoveries and, thus, welcome all follow-up studies that evaluate the reported cytotoxic effects of DT-061 on cancer cells, we are somewhat flabbergasted by the claim by Nilsson and colleagues that they could not confirm effects of the compound depended on PP2A. This is particularly surprising as we presented a series of orthogonal assays directed at defining the mode of action of DT-061, especially binding and stabilization of specific PP2A heterotrimer complexes, but not a single one of these experiments was performed precisely as described (Leonard et al 2020). Programs like The Open Science initiative emphasize the need to exactly reproduce experiments when testing for reproducibility. As such, the title of the Matters Arising opinion submitted by Vit et al is misleading, as no direct experiments were undertaken to show that DT-061 is unable to interact with PP2A.

Background: We developed a fluorophore-conjugated peptide agent, SBK4, that detects a tumor-specific proteolyzed form of the cell adhesion molecule, PTPmu, found in the tumor microenvironment. We previously demonstrated its tissue specific distribution in high-grade brain tumors. To extend those studies to other aggressive solid tumor types, we assessed the tissue distribution of PTPmu/SBK4 in a set of matched gynecologic cancer patient derived xenografts (PDXs) and primary patient tumors, as well as a limited cohort of tumors from gynecological cancer patients. PDXs isolated from the tissues of cancer patients have been shown to yield experimentally manipulatable models that replicate the clinical characteristics of individual patients’ tumors. In this study, gynecological cancer PDXs and patient biopsies were examined to determine if tumor-specific proteolyzed PTPmu was present. Methods: We used the peptide agent SBK4 conjugated to the fluorophore Texas Red (TR) to label tumor tissue microarrays (TMAs) containing patient and/or PDX samples from several high-grade gynecologic cancer types, and quantified the level of staining with Image J. In one TMA, we were able to directly compare the patient and the matched PDX tissue on the same slide. Results: While normal tissue had very little SBK4-TR staining, both primary tumor tissue and PDXs have higher labeling with SBK4-TR. Matched PDXs and patient samples from high-grade endometrial and ovarian cancers demonstrated higher levels of PTPmu by staining with SBK4 than normal tissue. Conclusion: In this sample set, all PDXs and high-grade ovarian cancer samples had increased labeling by SBK4-TR compared with the normal controls. Our results indicate that proteolyzed PTPmu and its novel peptide detection agent, SBK4, allow for the visualization of tumor-specific changes in cell adhesion molecules by tissue-based staining, providing a rationale for further development as an imaging agent in aggressive solid tumors, including gynecological cancers.

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.

Genomic instability (GI) 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 through the inhibition of RB1 and stimulator-of-interferon-genes (STING) to propagate cells with a high degree of GI. MiR-181a targeting of RB1 leads to profound nuclear defects and GI generating aberrant cytoplasmic DNA, however simultaneous miR-181a mediated inhibition of STING allows cells to bypass interferon mediated cell death. We also found that high miR-181a is associated with decreased IFNγ response and lymphocyte infiltration in patient tumors. DNA oncoviruses are the only known inhibitors of STING that allow for cellular transformation, thus, our findings are the first to identify a miRNA that can downregulate STING expression to suppress activation of intrinsic interferon signaling. This study introduces miR-181a as a putative biomarker and identifies the miR-181a-STING axis as a promising target for therapeutic exploitation., The majority of high grade serous ovarian cancers originate from fallopian tube secretory epithelial cells (FTSECs). Here the authors show that miR-181a drives oncogenic transformation in FTSECs through the cooperative inhibition of the tumor suppressor RB1 and of STING, resulting in genomic instability and suppression of intrinsic interferon signaling.

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.

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.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.

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.

CD55 is a membrane complement regulatory protein that attenuates complement-mediated cytotoxicity. Saygin et al. elucidate a new role for CD55 as a signaling hub for cancer stem cell self-renewal and cisplatin resistance pathways in endometrioid tumors and open a new line of research into chemotherapeutic-refractory cancers., Effective targeting of cancer stem cells (CSCs) requires neutralization of self-renewal and chemoresistance, but these phenotypes are often regulated by distinct molecular mechanisms. Here we report the ability to target both of these phenotypes via CD55, an intrinsic cell surface complement inhibitor, which was identified in a comparative analysis between CSCs and non-CSCs in endometrioid cancer models. In this context, CD55 functions in a complement-independent manner and required lipid raft localization for CSC maintenance and cisplatin resistance. CD55 regulated self-renewal and core pluripotency genes via ROR2/JNK signaling and in parallel cisplatin resistance via lymphocyte-specific protein tyrosine kinase (LCK) signaling, which induced DNA repair genes. Targeting LCK signaling via saracatinib, an inhibitor currently undergoing clinical evaluation, sensitized chemoresistant cells to cisplatin. Collectively, our findings identify CD55 as a unique signaling node that drives self-renewal and therapeutic resistance through a bifurcating signaling axis and provides an opportunity to target both signaling pathways in endometrioid tumors.

Targeted cancer therapies, which act on specific cancer-associated molecular targets, are predominantly inhibitors of oncogenic kinases. While these drugs have achieved some clinical success, the inactivation of kinase signaling via stimulation of endogenous phosphatases has received minimal attention as an alternative targeted approach. Here, we have demonstrated that activation of the tumor suppressor protein phosphatase 2A (PP2A), a negative regulator of multiple oncogenic signaling proteins, is a promising therapeutic approach for the treatment of cancers. Our group previously developed a series of orally bioavailable small molecule activators of PP2A, termed SMAPs. We now report that SMAP treatment inhibited the growth of KRAS-mutant lung cancers in mouse xenografts and transgenic models. Mechanistically, we found that SMAPs act by binding to the PP2A Aα scaffold subunit to drive conformational changes in PP2A. These results show that PP2A can be activated in cancer cells to inhibit proliferation. Our strategy of reactivating endogenous PP2A may be applicable to the treatment of other diseases and represents an advancement toward the development of small molecule activators of tumor suppressor proteins.

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

Characterizing the complex interplay of cellular processes in cancer would enable the discovery of key mechanisms underlying its development and progression. Published approaches to decipher driver mechanisms do not explicitly model tissue-specific changes in pathway networks and the regulatory disruptions related to genomic aberrations in cancers. We therefore developed InFlo, a novel systems biology approach for characterizing complex biological processes using a unique multidimensional framework integrating transcriptomic, genomic and/or epigenomic profiles for any given cancer sample. We show that InFlo robustly characterizes tissue-specific differences in activities of signalling networks on a genome scale using unique probabilistic models of molecular interactions on a per-sample basis. Using large-scale multi-omics cancer datasets, we show that InFlo exhibits higher sensitivity and specificity in detecting pathway networks associated with specific disease states when compared to published pathway network modelling approaches. Furthermore, InFlo's ability to infer the activity of unmeasured signalling network components was also validated using orthogonal gene expression signatures. We then evaluated multi-omics profiles of primary high-grade serous ovarian cancer tumours (N=357) to delineate mechanisms underlying resistance to frontline platinum-based chemotherapy. InFlo was the only algorithm to identify hyperactivation of the cAMP-CREB1 axis as a key mechanism associated with resistance to platinum-based therapy, a finding that we subsequently experimentally validated. We confirmed that inhibition of CREB1 phosphorylation potently sensitized resistant cells to platinum therapy and was effective in killing ovarian cancer stem cells that contribute to both platinum-resistance and tumour recurrence. Thus, we propose InFlo to be a scalable and widely applicable and robust integrative network modelling framework for the discovery of evidence-based biomarkers and therapeutic targets.

// 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.

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.

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.

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.

miRNAs are important regulators of diverse cellular processes including proliferation, apoptosis, and differentiation. In the context of bone marrow derived stromal cell and adipose derived stromal cell differentiation, miRNAs are established regulators of both differentiation or stemness depending on their target. Furthermore, miRNA dysregulation can play a key role in various disease states. Here we show that miR-181a is regulated in a circadian manner and is induced during both immortalized bone marrow derived stromal cell (iBMSC) as well as primary patient adipose derived stromal cell (PASC) adipogenesis. Enhanced expression of miR-181a in iBMSCs and PASCs produced a robust increase in adipogenesis through the direct targeting of the circadian factor period circadian regulator 3 (PER3). Furthermore, we show that knocking down endogenous miR-181a expression in iBMSC has a profound inhibitory effect on iBMSC adipogenesis through its regulation of PER3. Additionally, we found that miR-181a regulates the circadian dependency of the adipogenesis master regulator PPARγ. Taken together, our data identify a previously unknown functional link between miR-181a and the circadian machinery in immortalized bone marrow stromal cells and adipose derived stromal cells highlighting its importance in iBMSC and ASC adipogenesis and circadian biology.

The rat is the preferred model for toxicology studies, and it offers distinctive advantages over the mouse as a pre-clinical 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 toxicological testing and PK/PD studies in the same model animal. Here we created an immunodeficient rat with a functional deletion of the Rag2 gene, using genetically modified spermatogonial stem cells (SSCs). 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 27bp deletion mutation was identified and bred to homozygosity to produce the Sprague-Dawley Rag2 - Rag2(tm1Hera) (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. Additionally 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.

RNF126 is an E3 ubiquitin ligase. The deletion of RNF126 gene was observed in a wide range of human cancers and is correlated with improved disease-free and overall survival. These data highlights the clinical relevance of RNF126 in tumorigenesis and cancer therapy. However, the specific functions of RNF126 remain largely unknown. Homologous recombination (HR)-mediated DNA double-strand break repair is important for tumor suppression and cancer therapy resistance. Here, we demonstrate that RNF126 facilitates HR by promoting the expression of BRCA1, in a manner independent of its E3 ligase activity but depending on E2F1, a well-known transcription factor of BRCA1 promoter. In support of this result, RNF126 promotes transactivation of BRCA1 promoter by directly binding to E2F1. Most importantly, an RNF126 mutant lacking 11 amino acids that is responsible for the interaction with E2F1 has a dominant-negative effect on BRCA1 expression and HR by suppressing E2F1-mediated transactivation of BRCA1 promoter and blocking the enrichment of E2F1 on BRCA1 promoter. Lastly, RNF126 depletion leads to the increased sensitivity to ionizing radiation (IR) and poly (ADP-ribose) polymerase (PARP) inhibition. Collectively, our results suggest a novel role of RNF126 in promoting HR-mediated repair through positive regulation on BRCA1 expression by direct interaction with E2F1. This study not only offers novel insights into our current understanding of the biological functions of RNF126 but also provides a potential therapeutic target for cancer treatment.

// 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.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic tumors in human patients as well as nearisogenic pairs of high and low lung-metastatic osteosarcoma cells. We term these regions Metastatic Variant Enhancer Loci (Met-VELs). We demonstrate that these Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster non-randomly, indicating that activity of these enhancers and their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as F3. We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for anti-metastatic therapies.

Computation-based drug-repurposing/repositioning approaches 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 epithelial ovarian cancer (EOC) have not been undertaken. Here, we present a novel drug discovery strategy that combines a computational drug-repositioning system (DrugPredict) with biological testing in cell lines in order to rapidly identify novel drug candidates for EOC. DrugPredict exploited unique repositioning opportunities rendered by a vast amount of disease genomics, phenomics, drug treatment, and genetic pathway and uniquely revealed that non-steroidal anti-inflammatories (NSAIDs) rank just as high as currently used ovarian cancer drugs. As epidemiological studies have reported decreased incidence of ovarian cancer associated with regular intake of NSAIDs, we assessed whether NSAIDs could have chemoadjuvant applications in EOC and found that (i) NSAID Indomethacin induces robust cell death in primary patient-derived platinum-sensitive and platinum- resistant ovarian cancer cells and ovarian cancer stem cells and (ii) downregulation of β-catenin is partially driving effects of Indomethacin in cisplatin-resistant cells. In summary, we demonstrate that DrugPredict represents an innovative computational drug- discovery strategy to uncover drugs that are routinely used for other indications that could be effective in treating various cancers, thus introducing a potentially rapid and cost-effective translational opportunity. As NSAIDs are already in routine use in gynecological treatment regimens and have acceptable safety profile, our results will provide with a rationale for testing NSAIDs as potential chemoadjuvants in EOC patient trials.

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.

Host and Tumor Factor XII Drive Ovarian Cancer Maintenance and Progression Introduction . Epithelial ovarian cancer (EOC) is the leading cause of cancer death in women.Effective strategies to treat this disease are lacking due to the complexity of pathways involved and the multitude of cells that contribute to EOC biology. To this end, coagulation factor XII (FXII) and its receptor urokinase plasminogen activator receptor (uPAR) represent very promising therapeutic targets because i) uPAR is overexpressed in more than 90% of ovarian cancer patients, ii) FXII has been shown to be upregulated in the peritoneum of EOC patients and promotes EOC dissemination and iii) FXII-uPAR signal and upregulate key neutrophil functions, recently linked to tumor growth and metastasis. Through analysis of the TCGA ovarian cancer patient cohort, we have found that FXII and uPAR are co-expressed in ovarian tumors and their overexpression is associated with decreased overall survival (Fig 1A-B). Given that FXII and uPAR can be produced by both the EOC tumor and the host, the contribution of each of these compartments which has not been examined to date needs to be explored. Thus, we asked if the FXII-uPAR axis in neutrophils and EOC cells synergistically influences tumor biology. Methods - Results. We utilized a tissue microarray to determine FXII and uPAR expression. Both FXII and uPAR were co-expressed in all major histologic subtypes of EOC tumors but not in normal ovarian epithelium (Fig C-D). We adopted a composite expression score system and found significantly increased expression of FXII and uPAR in high grade tumors compared to low grade (grade 1-2) tumors, independently of tumor subtype and stage (Fig 1C-D). Given prior reports that neutrophils are enriched in the ovarian tumor microenvironment (TME) and our findings that FXII-uPAR are integral to neutrophil activation, tumors were also examined for their neutrophil content. Invariably, as tumor grade advanced, Neutrophil Elastase expression significantly increased (data not shown). We next asked if neutrophils contribute to EOC tumor progression or their recruitment merely correlates with advanced disease. We found that in the presence of neutrophils, EOC cells migrate significantly faster (p Next, in order to assess whether host FXII could contribute to EOC dissemination and progression we utilized WT, F12-/-, and uPAR deficient (Plaur-/-) mice for in vivo EOC tumor model. After orthotopic injection of ID8 EOC cells, WT mice exhibited faster rates of tumor development and ascitic fluid accumulation (13.4 ± 0.92 ml), relative to F12-/- (0.37 ± 0.26 ml) and Plaur-/- (0.5 ± 0.5 ml) mice (Fig 1G). Blinded examination of tumors showed significantly higher tumor burden in WT mice compared to F12-/-and Plaur-/- mice, with F12-/-mice exhibiting the highest degree of protection (Fig 1H). Conclusions . Our studies indicate three novel aspects: i) a direct crosstalk between ovarian cancer cells and neutrophils, that enhances tumor cell migration through FXII-uPAR signaling; ii) the presence of neutrophils in the TME induces EMT of cancer cells which is abolished when the FXII-uPAR interaction is inhibited and; iii) abrogation of the FXII-uPAR axis in EOC tumor cells and neutrophils, synergistically restricts the pro-invasive phenotype of ovarian tumor cells. Disclosures No relevant conflicts of interest to declare.

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.

EGFR activation is both a key molecular driver of disease progression and the target of a broad class of molecular agents designed to treat advanced cancer. Nevertheless, resistance develops through several mechanisms, including activation of AKT signaling. Though much is known about the specific molecular lesions conferring resistance to anti-EGFR–based therapies, additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. We identified a transcriptional network involving the tumor suppressors Krüppel-like factor 6 (KLF6) and forkhead box O1 (FOXO1) that negatively regulates activated EGFR signaling in both cell culture and in vivo models. Furthermore, the use of the FDA-approved drug trifluoperazine hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restored sensitivity to AKT-driven erlotinib resistance through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft models of lung adenocarcinoma. Combined, these findings define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs as capable of restoring chemosensitivity to anti-EGFR–based therapy for the treatment of metastatic lung adenocarcinoma.

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.