Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFβ) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFβ can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFβ treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFβ treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFβ-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFβ, in the clinical progression of NSCLC.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in 2-dimensional (2D) and 3-dimensional (3D) culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on Geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both Farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced more cell death in RH30 cells compared to C2C12 in 3D culture model with similar effects on autophagy flux as in 2D culture. Overall our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin myotoxicity is different in 2D versus 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatininduced cell death in myoblasts.

Poly(ADP‐ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage‐induced PARP1 activity. We used human triple‐negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage‐induced PARP1 activity, which was dependent on functional DNA‐binding AT‐hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

Introduction: Clear cell carcinoma of the ovary (CCC) is the 2nd most common ovarian cancer and is histologically and clinically distinct from other subtypes. Late stage CCC have a worse prognosis than other ovarian cancer histotypes as they are inherently resistant to the standard platinum/taxane chemotherapy. Deep endometriosis of the ovary, known as endometrioma or chocolate cyst, is the most common precursor for CCC. However, how transformation from endometriosis to CCC and, in particular, the important role of the hypoxic and ROS-rich microenvironment of endometriotic cysts are not well understood. As the molecular mechanisms pertinent to the genesis and progression of CCC are largely unknown, there are few, if any, therapeutic strategies for patients with advanced stage disease. Identifying factors that shape the development, progression and metastasis of CCC which can be targeted therapeutically could have tremendous potential to improve outcomes in this disease. Our recent findings identified cystathionine gamma-lyase (CTH), a key enzyme in the transsulfuration pathway, as a marker of Mullerian tract derived ciliated cells and CCC of both the ovary and uterus regardless of which mutations are present. Also, CTH is highly expressed both in CCC and the endometriosis adjacent to this cancer. Whether and how the transsulfuration pathway, notably CTH, enables CCC to adapt to the hostile microenvironment of an endometriotic cyst and ultimately to promote metastasis remain unanswered. Methods: We generated CTH knockout (KO) cells using CRISPR/Cas9. We assessed effects of CTH loss in vitro -under ambient and stress conditions- and in vivo on cell viability, cell proliferation, ROS levels, migration, invasion, and metastasis. Further, we used an organoid model system to assess the impact of CTH loss in primary endometrial cells on organoid growth and response to stress conditions including exposure to endometriotic cyst contents. Results: Our in-vitro, in-vivo data as well as data derived from the organoid modelling system show that CTH is critical for adaptive response to hypoxia. Further, it underpins the growth of CCC cells in-vitro and the growth of CTH in a mouse model of CCC. These data highly indicate that expression of the transsulfuration pathway enzyme CTH enables cells to survive in endometriotic cysts then upon transformation both marks CCC and potentiates tumor progression and metastasis. Conclusion: Targeting CTH in CCC and potentially other cancers might represent a novel and impactful therapeutic approach. Citation Format: Amal M. El-Naggar, Yuchen Ding, Genny Trigo-Gonzalez, Lucy Li, Shary Chen, Busra Turgu, Forouh Kalantari, Rodrigo Vallejos, Kiran Parmar, Cindy Shen, Gian Luca Negri, Paul Yong, Gregg Morin, David G. Huntsman. On the verge of metastasis: CTH defines a cell-state-dependent adaptive response to microenvironmental stresses [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 2482.

Introduction: High-grade serous ovarian cancer (HGSOC) is driven by loss of TP53 and genome instability . Despite the recent success of PARP inhibitors, advanced BRCA mutant and homologous recombination repair deficient (HRD) HGSOC, chemotherapy remains the first line treatment. Little is known about how chemotherapy exposure alters tumor heterogeneity and subsequent response to targeted therapies. Certain mutations may survive the exposure of chemotherapy better than others. The question of whether targeted therapies should be given before or after response to chemotherapy also remains unanswered. We use the principles of natural selection to investigate how HGSOC evolves over time and selection operates on clones in the context of cytotoxic/non-cytotoxic combination therapies, and what changes in genomes/transcriptomes at the single cell level drive tumor progression. Methods: Cell lines ID8 Trp53 −/−; Brca1 −/− and WT were used to represent defective/proficient homologous recombination were given intraperitoneally to C57B6 mice to develop HGSOC models. Transfected Luciferase expression in the cells used to monitor tumor response to olaparib (Ola)+/- Bevacizumab (BEV-various doses) +/- Atezolizumab (ATz) combinations by bioluminescence imaging (BLI). Ascitic fluid and mouse organs were harvested for the evidence of seeding and identification of biomarkers. In addition, 10 treatment naïve HGSOC PDX were developed in immunodeficient mice and treated with either cisplatin or olaparib. Single-cell whole-genome sequencing (scWGS) was performed using direct library preparation (DLP+). Hierarchical clustering and Sitka are used to identify the clonal structure of each condition following treatment. Phylogenetic tree was computed using copy number data. Results: In the BRCA WT HRp study group, the greatest response was seen in the triplet Ola+BEV+ATz combinations and interestingly no significant differences were observed using a lower dose of BEV. Furthermore, from scWGS data of PDX passages we captured initial clonal heterogeneity leading to emergent clones. We found evolving copy number changes on chromosome 19, 8, 3 and loss of heterozygosity of TP53. Conclusion: The triplet combination of low-dose-intensity bevacizumab with other non-cytotoxic drugs was an effective regimen for BRCA WT syngeneic mouse tumors. In HGSOC PDX, we are able to capture diversification between HGSOC PDX passages-specifically after drug exposures. Citation Format: Farhia Kabeer, Goldman Lam, Naila Adam, Maxwell Douglas, Amal El-Naggar, Forouh Kalantari, Mengke Han, Vinci Au, Michael Van Vliet, Cindy Shen, Sean Beaty, Daniel Lai, Andy Mungall, Richard Moore, Sam Aparicio, Andrew Roth, David Huntsman, Yvette Drew. The evolution of high grade serous ovarian cancer under the pressure of non-cytotoxic and cytotoxic treatment [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 2182.

Introduction Clear cell ovarian cancer (CCC) accounts for 5-11% of ovarian cancers in North America with a higher frequency reported in cohorts from Japan. CCC usually arises directly from endometriosis, which is associated with oxidative stress. The latter plays a critical role in the pathogenesis of both endometriosis and CCC. CCC is inherently resistant to standard chemotherapies, and the outcomes for patients with advanced stage CCC have not changed in several decades. Therefore, there is an urgent need for new treatment options for advanced CCC. CCC possess a distinct genetic profile; >50% harbour ARID1A mutations typically resulting in loss of protein function and often with co-occurrence of PIK3CA mutation or gene amplification leading to PI3K/AKT pathway hyper-activation. ARID1A mutations leads to accumulation of ROS, decrease in GSH due to downregulation of SLC7A11 and related reduction in the GSH precursor cystine thereby rendering cancer cells highly dependent on oxidative phosphorylation. EO3001 is a small molecule drug candidate with selective activity against ARID1A-deficient cell lines in vitro. It has been shown that EO3001 directly binds and inhibits FDX-1 function to block iron-sulfur cluster formation in complex I, a critical component of the mitochondrial electron transport chain. Iron-sulfur clusters play a critical role in the oxidation-reduction reactions of electron transport in mitochondria relied on by cancer cells that have made an adaptive shift from glycolysis to high mitochondrial dependence. Complex I plays a role in redox control and the biosynthesis of macromolecules and nucleic acids necessary for cell proliferation. It is suggested that these complex I-dependent events contribute to tumor formation, resistance to cell death, and metastasis of cancer cells in part by causing an increase in ROS levels Methods We generated isogenic ovarian cancer cell lines (RMG-1 and OVCA438, -/+ ARID1A loss) using CRISPR/Cas9. We will assess therapeutic effects of EO3001 on cells’ tumorigenic potential in vitro -under ambient and stress conditions, notably endometriotic cyst content which derives malignant transformation, and in vivo on cell viability, cell proliferation, ROS levels, migration, invasion, and metastasis. We will use the organoids modeling system -using primary endometrial cells harboring ARID1A mutations- to assess the impact of EO3001 on organoid growth and response to stress conditions and evaluate the effect of EO3001 on cancer metastesis by the ex vivo pulmonary metastasis assay (PuMA). Conclusions Exploiting the vulnerability in reliance on OXPHOS in ARID1A-deficient CCC using EO3001 might represent a promising strategy for the treatment of these patients as well as patients harboring other ARID1A-deficient malignancies. Citation Format: Amal M. EL-Naggar, Yuchen Ding, Lucy Li, Forouh Kalantari, Jeffrey Bacha, Dennis Brown, David Huntsman. Investigating the therapeutic efficacy of EO3001 in clear cell carcinoma of the ovary. [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 4967.

INTRODUCTION: Ovarian cancer is the 5th deadliest cancer found in women and is the deadliest involving the gynecological tract. Most epithelial ovarian cancers have extra-ovarian origins and can be stratified into various histotypes: high and low-grade serous (HGS and LGS), endometrioid (ENOC), clear cell (CCOC), and mucinous – each of which are proposed to have distinct precursor lesions. We present organoids as a useful model to study precursor lesions and the process of tumorigenesis in epithelial ovarian carcinomas. Organoids recapitulate the in vivo growth microenvironment and are useful to study the mechanisms of tumorigenesis from healthy cells. We have previously proposed that ENOC arise from the secretory cell lineage, while CCOC originate from the ciliated cell lineage, and organoids are an ideal model to examine in greater depth the impact of mutation on specific cell populations, such as ciliated cells. METHODS: Surgical fallopian tube and endometrial tissues, removed for non-cancer reasons, were cultured in 2D followed by plating into Matrigel. Matrigel cultures were supplemented with media containing stem/progenitor differentiation factors promoting organoid growth. To study the effect of mutations often found in ovarian cancers on organoid growth and development, gene knockouts were produced using CRISPR lentiviruses on cells prior to Matrigel culture. Lentiviral transductions were optimized for organoid formation and for minimizing invasiveness accrued on cells. CRISPR gRNA constructs were validated by Western Blot and qPCR. Organoids containing knockouts of p53, BRCA1 and BRCA2 were used to model precursor lesions of HGS, whereas ARID1A knockouts and an inducible PIK3CA activating mutations were used to model CCOC. To gain further insight into ciliated cells of the endometrium, organoids were treated with the notch inhibitor-DBZ to drive differentiation of cells towards a ciliated cell lineage. We analyzed organoids by single-cell RNA sequencing (scRNA-seq), immunohistochemistry (IHC), and immunofluorescence staining (IF). Single cells were derived by purifying the organoids from Matrigel followed by a chemical and physical digestion. scRNA-seq was performed utilizing the 10X Genomics Platform and analyzed by in-house bioinformaticians. Bioinformatic analyses included stringent QC to remove low-quality and dead cells, before applying unsupervised learning algorithms like PCA and Gaussian mixture modeling as well as differential expression analysis to understand both how samples relate to each other and cell types discovered within each sample. RESULTS: We successfully recapitulated the histology observed in tissues by growing endometrial and fallopian tube organoids. The notch inhibitor, DBZ forced ciliated cell differentiation, as observed by IHC, IF and scRNA-seq. scRNA-seq clustering of DBZ-treated organoid cultures revealed a possible intermediary state between progenitor and ciliated cells. Initial IHC and IF analyses of CRISPR-mediated organoids reveal successful gene manipulation. CONCLUSIONS: Organoid cultures present as a powerful method for modelling precursor lesions; they can be readily manipulated genetically and with rapid turnaround compared to conventional mouse models. Organoids are also amenable to sequencing at single-cell resolution. The ability to model ovarian cancers with permanent knockouts in human tissue serves as a necessary link between animal models and human therapy. Citation Format: Germain C. Ho, Dawn R. Cochrane, Evan W. Gibbard, Kieran Campbell, Basile Tessier-Cloutier, Kendall Greening, Forouh Kalantari, Genny Trigo-Gonzalez, Yemin Wang, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. MODELS AND ANALYTIC TECHNIQUES OF MULLERIAN TISSUE-DERIVED ORGANOIDS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-030.