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Carbon monoxide (CO) has long been considered a toxic gas but is now a recognized bioactive gasotransmitter with potent immunomodulatory effects. Although inhaled CO is currently under investigation for use in patients with lung disease, this mode of administration can present clinical challenges. The capacity to deliver CO directly and safely to the gastrointestinal (GI) tract could transform the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, inspired by molecular gastronomy techniques, we have developed a family of gas-entrapping materials (GEMs) for delivery of CO to the GI tract. We show highly tunable and potent delivery of CO, achieving clinically relevant CO concentrations in vivo in rodent and swine models. To support the potential range of applications of foam GEMs, we evaluated the system in three distinct disease models. We show that a GEM containing CO dose-dependently reduced acetaminophen-induced hepatocellular injury, dampened colitis-associated inflammation and oxidative tissue injury, and mitigated radiation-induced gut epithelial damage in rodents. Collectively, foam GEMs have potential paradigm-shifting implications for the safe therapeutic use of CO across a range of indications.

Carbon monoxide (CO) is one of the most significant signal molecules and plays an important role in regulating human physiological and pathological processes. In this study, a novel Pd-based complex (Pd-BNP-OH) was developed for endogenous CO detection. The structure and morphology of Pd-BNP-OH was characterized by SEM, XPS, and NMR analyses. When Pd-BNP-OH was reacted with CO, a strong fluorescence enhancement at 510 nm was observed. In addition, Pd-BNP-OH exhibited high stability and selectivity toward CO in PBS buffer. In biological experiments, Pd-BNP-OH exhibited little cytotoxicity in cellular environment, and a bright fluorescence turn on was observed in the presence of exogenous CO and endogenous generated CO. The probe was then applied for CO detection in live zebrafish by both one-photon and two-photon excitation. Significantly, Pd-BNP-OH has excellent two-photon property, controllable structure and high biocompatibility. These features enable the probe to detect endogenously generated carbon monoxide in live organisms successfully.

Simple Summary In the conventional treatment of gynecologic malignancies, most patients receive similar ‘one-size-fits-all’ treatment. However, it is increasingly clear that standard therapies do not work in every patient, and it would be very helpful to have pretreatment predictive assays to provide more personalized regimens. In this study, we describe the routine, successful establishment of patient-derived organoid models (PDOs) of endometrial and ovarian cancer tissues from consenting patients and provide an example of how information from drug screening in PDOs may be a useful predictor of patient response to therapy. Abstract Developing reliable experimental models that can predict clinical response before treating the patient is a high priority in gynecologic cancer research, especially in advanced or recurrent endometrial and ovarian cancers. Patient-derived organoids (PDOs) represent such an opportunity. Herein, we describe our successful creation of 43 tumor organoid cultures and nine adjacent normal tissue organoid cultures derived from patients with endometrial or ovarian cancer. From an initial set of 45 tumor tissues and seven ascites fluid samples harvested at surgery, 83% grew as organoids. Drug sensitivity testing and organoid cell viability assays were performed in 19 PDOs, a process that was accomplished within seven days of obtaining the initial surgical tumor sample. Sufficient numbers of cells were obtained to facilitate testing of the most commonly used agents for ovarian and endometrial cancer. The models reflected a range of sensitivity to platinum-containing chemotherapy as well as other relevant agents. One PDO from a patient treated prior to surgery with neoadjuvant trastuzumab successfully predicted the patient’s postoperative chemotherapy and trastuzumab resistance. In addition, the PDO drug sensitivity assay identified alternative treatment options that are currently used in the second-line setting. Our findings suggest that PDOs could be used as a preclinical platform for personalized cancer therapy for gynecologic cancer patients.

Objectives: Antiangiogenics have become a cornerstone of treatment in advanced stage and recurrent high grade serous ovarian cancer. Both cediranib (ICON6) and bevacizumab (ICON7, GOG218) have demonstrated benefit as a maintenance treatment for ovarian cancer patients in clinical trials. Investigators have also explored their role in advanced stage and recurrent endometrial carcinoma (MITO-END 2, Rose 2017, Aghajanian 2011). There have been no head to head trials in gynecologic cancers comparing cediranib to bevacizumab, although in colorectal carcinoma, phase III data suggests equivalent efficacy (HORIZON III). We hypothesize that these drugs, although both categorized as anti-angiogenics, induce unique cellular responses and sought to explore the varying impact of each agent on cell signaling using established serous cell models. Methods: CAOV3 ovarian cancer cells and Hec50 endometrial cancer cells were exposed to 1mM bevacizumab or cediranib for 24 hours. Cell lysates were analyzed in duplicate using an antibody-based phosphoproteomic array of 875 phosphoproteins supplied by Kinexus Bioinformatics (Vancouver, BC). Priority signaling effects were defined for each drug and in each cell line as those with a significant (>75%) change from control with an acceptable sum of error ( Results: Bevacizumab yielded 13 total leads, and cediranib yielded 9 in CAOV3 ovarian cancer cells (Table 1). Both agents significantly enhanced signaling events on the following epitopes: ABl, EZH2, HGK (2 sites), and IGF1R, indicating that these may be some of the core cellular effects of anti-angiogenic agents. Interestingly, all common leads induced by both agents in CAOV3 cells represent potential pro-oncogenic effects. In Hec50 cells (Table 1), both cediranib and bevacizumab treatment induced a unique set of phosphoproteins compared to the effects of these agents in CAOV3 cells, yet common findings were the phosphorylation of IGFR1 by bevacizumab and the activation of alternative MAP kinases in both cell lines. Download : Download high-res image (128KB) Download : Download full-size image Conclusions: Angiogenesis plays a crucial role in tumor development and metastasis. Blocking angiogenesis using monoclonal antibodies such as bevacizumab or tyrosine kinase inhibitors such as cediranib is a strategy that is proving to be helpful in the care of patients with gynecologic malignancies. These molecular agents have both on-target, therapeutic effects but also may induce pathways of resistance. In these comprehensive phosphoproteomic mapping studies, we noted the activation of signaling pathways which were cell and drug specific. One consistent finding was the enhanced phosphorylation of IGFR1 and alternative MAP kinases that may portend eventual drug resistance.

Objective Platinum compounds have been widely used as a primary treatment for many types of cancer. However, resistance is the major cause of therapeutic failure for patients with metastatic or recurrent disease, thus highlighting the need to identify novel factors driving resistance to Platinum compounds. Metadherin (MTDH, also known as AEG-1 and LYRIC), located in a frequently amplified region of chromosome 8, has been consistently associated with resistance to chemotherapeutic agents, though the precise mechanisms remain incompletely defined. Methods The mRNA of FANCD2 and FANCI was pulled down by RNA-binding protein immunoprecipitation. Pristimerin-loaded nanoparticles were prepared using the nanoprecipitation method. Immunocompromised mice bearing patient-derived xenograft tumors were treated with pristimerin-loaded nanoparticles, cisplatin and a combination of the two. Results MTDH, through its recently discovered role as an RNA binding protein, regulates expression of FANCD2 and FANCI, two components of the Fanconi anemia complementation group (FA) that play critical roles in interstrand crosslink damage induced by platinum compounds. Pristimerin, a quinonemethide triterpenoid extract from members of the Celastraceae family used to treat inflammation in traditional Chinese medicine, significantly decreased MTDH, FANCD2 and FANCI levels in cancer cells, thereby restoring sensitivity to platinum-based chemotherapy. Using a patient-derived xenograft model of endometrial cancer, we discovered that treatment with pristimerin in a novel nanoparticle formulation markedly inhibited tumor growth when combined with cisplatin. Conclusions MTDH is involved in post-transcriptional regulation of FANCD2 and FANCI. Pristimerin can increase sensitivity to platinum-based agents in tumors with MTDH overexpression by inhibiting the FA pathway.

Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death driven by lipid hydroperoxides within biological membranes. Although therapy-resistant mesenchymal-high cancers are particularly vulnerable to ferroptosis inducers, especially phospholipid glutathione peroxidase 4 (GPx4) inhibitors, the underlying mechanism is yet to be deciphered. As such, the full application of GPx4 inhibitors in cancer therapy remains challenging. Here we demonstrate that metadherin (MTDH) confers a therapy-resistant mesenchymal-high cell state and enhanced sensitivity to inducers of ferroptosis. Mechanistically, MTDH inhibited GPx4, as well as the solute carrier family 3 member 2 (SLC3A2, a system Xc− heterodimerization partner), at both the messenger RNA and protein levels. Our metabolomic studies demonstrated that MTDH reduced intracellular cysteine, but increased glutamate levels, ultimately decreasing levels of glutathione and setting the stage for increased vulnerability to ferroptosis. Finally, we observed an enhanced antitumor effect when we combined various ferroptosis inducers both in vitro and in vivo; the level of MTDH correlated with the ferroptotic effect. We have demonstrated for the first time that MTDH enhances the vulnerability of cancer cells to ferroptosis and may serve as a therapeutic biomarker for future ferroptosis-centered cancer therapy.

Objectives: Though many ovarian cancer patients respond to primary treatment, a significant portion will not respond and are considered ‘platinum resistant/refractory.’ Such patients experience a significantly worse prognosis relative to those who are ‘platinum sensitive.’ Pre-treatment prediction of platinum sensitivity has eluded scientists, which forces clinicians to undergo a ‘trial and error’ method of treatment by which resistant patients are forced to try and fail a futile line of therapy. Patient-derived organoid (PDO) cultures represent a potentially powerful functional model which preserves tumor heterogeneity (Hill et al, 2018). Here, we describe the successful culture of 4 PDOs from patients with advanced stage ovarian carcinoma and their correlation with clinical treatment outcome. Methods: Fresh tissue was obtained from the patients’ debulking surgery. It was digested, and a cell pellet was created. The pellet was washed several times, re-suspended in Matrigel, plated, and grown at 37oC over several days. PDOs were treated with standard doses of carboplatin (1 µM) and paclitaxel (10 nM). The percent of viable cells at 72 hours was measured using the CellTiter-Glo 3D platform (Promega, Madison, WI). The patients’ clinical charts were then reviewed 6 months after the completion of primary chemotherapy to determine clinical platinum sensitivity as defined by clinical parameters. Results: PDOs were successfully obtained from each of the four patients with advanced stage ovarian carcinoma. Three patients had primary disease, and 1 patient was being treated for a recurrence. The patient with recurrent disease received chemotherapy immediately prior to her debulking surgery, and the rest underwent primary debulking surgery with chemotherapy to follow. Three patients had high grade serous histology, and 1 patient had low grade serous histology. Three patients ultimately had platinum resistant disease, and their PDOs all demonstrated >50% cell viability in response to carboplatin and paclitaxel with 2 resistant PDOs demonstrating a remarkable 90% and 95% cell viability. The PDO from the patient with platinum sensitive disease by clinical criteria demonstrated 49% cell viability in response to treatment (Table 1). Download : Download high-res image (123KB) Download : Download full-size image Conclusions: We describe the successful creation of PDOs from fresh tissue obtained from debulking surgery in advanced stage ovarian cancer patients. If we use ≥50% cell viability as our initial, preliminary sensitivity threshold, results from the PDO drug sensitivity assay correlate with clinical platinum sensitivity in this pilot feasibility. We propose to confirm the predictability of drug sensitivity thresholds in future, larger studies using PDOs. Currently, there is no standardized drug sensitivity assay or response parameters defined as predictable for PDOs. Such work will be integral in the formation of a functional model of drug sensitivity, which can be used in the clinic to predict platinum sensitivity without requiring a patient to try and fail futile treatments.

// Jianling Bi 1 , Bo Hu 1 , Jing Wang 1 , Xing Liu 1 , Jinsong Zheng 1 , Ding Wang 1 and Wuhan Xiao 1, 2 1 The Key Laboratory of Aquatic Biodiversity and Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China 2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, P. R. China Correspondence to: Wuhan Xiao, email: w-xiao@ihb.ac.cn Ding Wang, email: wangd@ihb.ac.cn Keywords: beluga whale, cetaceans, hypoxia, HIF-2α, VHL Received: October 09, 2016 Accepted: January 09, 2017 Published: February 02, 2017 ABSTRACT Aquatic mammals, such as cetaceans experience various depths, with accordingly diverse oxygenation, thus, cetaceans have developed adaptations for hypoxia, but mechanisms underlying this tolerance to low oxygen are unclear. Here we analyzed VHL and HIF-2α, in the hypoxia signaling pathway. Variations in VHL are greater than HIF-2α between cetaceans and terrestrial mammals, and beluga whale VHL (BW-VHL) promotes HIF-2α degradation under hypoxia. BW-VHL catalyzes BW-HIF-2α to form K48-linked poly-ubiquitin chains mainly at the lysine 429 of BW-HIF-2α (K429) and induces BW-HIF-2α for proteasomal degradation. W100 within BW-VHL is a key site for BW-VHL functionally and BW-VHL enhances transcriptional activity of BW-HIF-2α under hypoxia. Our data therefore reveal that BW-VHL has a unique function that may contribute to hypoxic adaptation.

Patient-derived organoids (PDOs) are a promising model for personalized cancer treatment. Accumulating evidence indicates that PDOs can predict clinical outcomes in patients. A handful studies in several cancer types have established that PDOs can recapitulate both histological and genomic features of the lesion from which they were derived. Additionally, PDOs can grow with high efficiency in a short time, enabling prediction of responsiveness to chemotherapy and development of more targeted therapies for patients. Our long-term goal is to create a PDO biorepository comprising all major histopathological subtypes of endometrial cancer, perform drug screening on these PDOs, and identify novel single agent and combinatorial regimens to advance into clinical trials. Herein we describe progress to date on generation of endometrial PDOs and drug screening to identify potential therapeutic options. First, we created PDO cultures from seven patients with different histological subtypes of endometrial cancer, including early stage/grade endometrioid adenocarcinoma and high-grade serous carcinoma. Next, we exposed each PDO to a panel of 23 chemotherapeutic agents and targeted drugs. Surprisingly, despite a divergent response to standard chemotherapy (platinum and taxane compounds), all PDOs showed high responsiveness to the combination of a histone deacetylase (HDAC) inhibitor and a proteasome inhibitor. Specifically, we achieved nearly complete cell killing within 72 hrs of exposure of organoid cultures to HDAC and proteasome inhibitors, with concentrations in the low nanomolar range. Similar results were achieved with different proteasome inhibitors (bortezomib and ixazomib) and HDAC inhibitors (romidepsin and belinostat). At present, these classes of agents have primarily been studied in multiple myeloma, though multiple clinical trials of combined proteasome and HDAC inhibitor therapy are active in advanced solid tumors and lymphomas. HDAC inhibitors maintain the acetylation of histones and thereby increase the expression of genes associated with apoptosis and cell cycle arrest. Proteasome inhibitors prevent proteasomal degradation, leading to upregulation of proapoptotic protein expression, cell cycle arrest, and ER stress, downregulation of angiogenesis and the pro-inflammatory protein NF-κB, and ROS generation. Mechanistically, HDAC inhibitors and proteasome inhibitors promote dual proteasome and aggresome blockage and induce apoptosis due to the accumulation of misfolded proteins. Our findings of profound tumor cell death in multiple different PDOs serve as the foundation for a future clinical trial using the HDAC inhibitor ixazomib and proteasome inhibitor romidepsin in advanced and recurrent endometrial cancers as well as high-grade serous ovarian cancer, which shares histologic and molecular features. Concomitantly, we are investigating the in vitro and in vivo effects of the combination on the induction of apoptosis and endometrial tumor regression. Citation Format: Jianling Bi, Kristina W. Thiel, Eric J. Devor, Andreea M. Newtson, Yuping Zhang, Kimberly K. Leslie. Combined histone deacetylase and proteasome inhibition in patient-derived endometrial cancer organoid models promotes massive cell death [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO039.

Angiogenesis plays a crucial role in tumor development and metastasis, and many cancer cells upregulate vascular endothelial growth factor-A (VEGF-A) expression to promote angiogenesis. Several clinical trials of anti-angiogenic agents have been conducted in advanced and recurrent endometrial cancer, but only bevacizumab and cediranib have demonstrated activity as single agents. Bevacizumab is a monoclonal antibody against VEGF-A, whereas cediranib is a small molecule angiokinase inhibitor. However, subsequent studies of bevacizumab+chemotherapy failed to improve outcomes compared to chemotherapy alone. We recently demonstrated that chemotherapy plus nintedanib, another angiokinase inhibitor, promotes catastrophic cell death in a xenograft model of endometrial cancer (Ebeid, et al., Nat Nanotech 2019). This effect was specific to cells with loss of function, i.e., null mutations in TP53. Here we compared the efficacy of cediranib vs. bevacizumab in p53-null gynecologic cancer models and determined the mechanism for differential sensitivity. We first performed a phosphoproteomic array of 875 phosphoproteins to define the signaling changes related to bevacizumab vs. cediranib in two p53-null cell gynecologic cancer cell lines. Several signaling events were similar between bevacizumab and cediranib, including phosphorylation of IGF1R, Abl, p70S6K and proteins in the ERK/MAPK and Wnt signaling pathways. We next tested the impact of the anti- angiogenic agents on cell viability using cancer cell lines and over 20 patient-derived organoid cultures of endometrial or ovarian cancer. Neither bevacizumab nor cediranib alone had a notable effect on cell viability, even at 1-10 µM concentrations. By contrast, cediranib but not bevacizumab promoted marked cell death when combined with chemotherapy. This effect was most pronounced in p53-null models. Cell cycle analysis demonstrated an accumulation in mitosis after treatment with cediranib+chemotherapy, consistent with abrogation of the G2/M checkpoint and subsequent mitotic catastrophe. Molecular analysis of key controllers of the G2/M cell cycle checkpoint confirmed its abrogation. Unexpectedly, the endometrial cancer cell lines were unresponsive to stimulation with VEGF-A, the target of bevacizumab. This was confirmed by the lack of ERK phosphorylation, a downstream signaling event expected after VEGF-A treatment, and was potentially caused low expression of VEGFR1 and 2. Based on these data, we conclude that an anti-angiogenic tyrosine kinase inhibitor such as cediranib has the potential to be superior to bevacizumab in combination with chemotherapy. We hypothesize that the mechanism relates to cediranib inhibition of the tumor vasculature as well as its ability to optimize the impact of chemotherapeutic agents during the cell cycle. Future clinical studies should test this hypothesis by studying the combination of standard chemotherapy with cediranib in advanced and recurrent endometrial cancer. Citation Format: Jianling Bi, Andreea M. Newtson, Eric J. Devor, Yuping Zhang, Kristina W. Thiel, Kimberly K. Leslie. Anti-angiogenic tyrosine kinase inhibitor cediranib is superior to bevacizumab in endometrial cancer due to differential effects on cell cycle regulation [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO046.

Advanced stage serous carcinoma of the uterus typically portends a poor prognosis. There is an urgent need to augment clinical trial data with different methods of treatment evaluation before a patient is made to try and potentially fail a therapy. Patient derived organoids (PDOs) may represent such an opportunity. We describe the successful culture and drug sensitivity testing of two PDOs from patients with advanced stage serous carcinoma of the uterus who received neoadjuvant chemotherapy. Clinical response to treatment was compared with the organoid response to the same regimens. Drug sensitivity data are reported as percentage of residual cell viability 48 hours after one standard treatment dose, in which 100% viability indicates no response and 0% viability reflects complete cell death. We hypothesized that significant cell killing, i.e. a low cell viability in our assay, would be required to achieve clinical drug sensitivity in patients. Both patients were clinically platinum resistant. Patient 1 had a large burden of disease at the time of presentation, including skeletal and liver metastasis. Though she initially partially responded to triplet therapy of paclitaxel, carboplatin and bevacizumab, she ultimately had persistent unresectable disease after 6 cycles of therapy. The percent residual, post-drug cell viability for PDO-1 was 45% in response to paclitaxel, carboplatin and bevacizumab combinatorial treatment, 35% in response to paclitaxel and carboplatin, and 100% in response to single agent bevacizumab. Patient 2 received paclitaxel, carboplatin and trastuzumab because her tumor was positive for HER2neu. Though she also had an initial response to treatment, she experienced a recurrent malignant pleural effusion 3 months after completing platinum-containing triplet (trastuzumab was continued as a maintenance therapy). The percent residual cell viability for PDO-2 was 51% in response to the combination of paclitaxel and carboplatin. Previous investigators have identified PDOs as a potentially powerful functional model for treatment response. We confirm that it is feasible to establish organoids and to rapidly test agents for cell sensitivity even prior to the first post-surgical chemotherapeutic treatment cycle. For both patients, the PDO models were only moderately sensitive to the agents used for therapy, and the lack of long-term benefit achieved for these patients may have been suggested a priori by the PDO models. However, further studies are necessary to establish a threshold percent value of post-treatment cell viability that is predictive of clinical patient response. We hypothesize that such a threshold appears to be significantly below 50% from these two initial cases. Citation Format: Andreea M. Newtson, Jianling Bi, Yuping Zhang, Eric Devor, Kristina Thiel, Kimberly K. Leslie. Development of patient derived organoid cultures of two advanced stage serous carcinomas of the uterus [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PO025.

Tumor suppressor p53 is responsible for enforcing cell cycle checkpoints at G1/S and G2/M in response to DNA damage, thereby allowing both normal and tumor cells to repair DNA before entering S and M. However, tumor cells with absent or mutated p53 are able to activate alternative signaling pathways that maintain the G2/M checkpoint, which becomes uniquely critical for the survival of such tumor cells. We hypothesized that abrogation of the G2 checkpoint might preferentially sensitize p53-defective tumor cells to DNA-damaging agents and spare normal cells with intact p53 function. The tyrosine kinase WEE1 regulates cdc2 activity at the G2/M checkpoint and prevents entry into mitosis in response to DNA damage or stalled DNA replication. AZD1775 is a WEE1 inhibitor that overrides and opens the G2/M checkpoint by preventing WEE1-mediated phosphorylation of cdc2 at tyrosine 15. In this study, we assessed the effect of AZD1775 on endometrial and ovarian cancer cells in the presence of two DNA damaging agents, the PARP1 inhibitor, olaparib, and the chemotherapeutic agent, gemcitabine. We show that AZD1775 alone is effective as a therapeutic agent against some p53 mutated cell models. Moreover, the combination of AZD1775 with olaparib or gemcitabine is synergistic in cells with mutant p53 and constitutes a new approach that should be considered in the treatment of advanced and recurrent gynecologic cancer.

Mutations in the “guardian of the genome” TP53 predominate in solid tumors. In addition to loss of tumor suppressor activity, a specific subset of missense mutations confers additional oncogenic properties. These “gain-of-function” (GOF) mutations portend poor prognosis across cancer types regardless of treatment. Our objective in this study was to identify novel therapeutic opportunities to overcome the deleterious effects of GOF TP53 mutants. Using gynecologic cancer cell lines with known TP53 mutational status, we established that treatment with a proteasome inhibitor induced cell death in cells with two recurrent GOF TP53 mutations (R175H and R248Q), and addition of a histone deacetylase inhibitor (HDACi) enhanced this effect. By contrast, p53-null cancer cells were relatively resistant to the combination. Towards understanding the mechanism, we found that proteasome inhibition promotes apoptosis of cells with TP53 GOF mutations, potentially through induction of the unfolded protein response. In line with the reported hyperstabilization of GOF p53 protein, cells treated with HDACi exhibited reduced levels of p53 protein. Together, these data form the basis for future clinical studies examining therapeutic efficacy in a preselected patient population with GOF TP53 mutations.

Cetaceans have evolved many behavioral and biological adaptations to allow them to survive in aquatic environments. Because of diving and breath-holding behaviors, cetaceans experience wide fluctuations in oxygen tension. However, the molecular basis behind their adaptation to a wide range of diving depths and durations remains unknown. Hypoxia-inducible factor (HIF) is a crucial regulator of cellular and systemic responses to low oxygen levels. In this study, the HIF-1α gene was cloned from the sperm whale (Physeter macrocephalus), the beluga whale (Delphinapterus leucas), and the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis). HIF-1α from the deep-diving sperm whale degraded rapidly under normoxia but remained stable during hypoxia compared with HIF-1α from the beluga whale and the Yangtze finless porpoise. Sperm whale HIF-1α also responded to reoxygenation significantly faster and exerted more potent effects on the transcriptional activity of some gene promoters compared with HIF-1α from the beluga whale and the Yangtze finless porpoise. Mutations in HIF-1α were detected between the Yangtze finless porpoise and the sperm whale, which may determine their functional differences. The results suggest that the response of sperm whale HIF-1α to different oxygen tensions is more variable than that of Yangtze finless porpoise or beluga whale HIF-1α, indicating that the rate of degradation of HIF-1α may be more variable in long-duration divers than in short-duration diver cetaceans. Because of the lack of whale models and whale cell lines, all experiments were conducted in vitro using human embryonic kidney 293T cells.

Mutations in the “guardian of the genome” TP53 predominate in solid tumors. In addition to loss of tumor suppressor activity, a specific subset of missense mutations confers additional oncogenic properties. These “gain-of-function” (GOF) mutations portend poor prognosis across cancer types regardless of treatment. Our objective in this study was to identify novel therapeutic opportunities to overcome the deleterious effects of GOF TP53 mutants. Using gynecologic cancer cell lines with known TP53 mutational status, we established that treatment with a proteasome inhibitor induced cell death in cells with two recurrent GOF TP53 mutations (R175H and R248Q), and addition of a histone deacetylase inhibitor (HDACi) enhanced this effect. By contrast, p53-null cancer cells were relatively resistant to the combination. Proteasome inhibition promoted apoptosis of cells with TP53 GOF mutations, potentially through induction of the unfolded protein response. In line with the reported hyperstabilization of GOF p53 protein, cells treated with HDACi exhibited reduced levels of p53 protein. Together, these data form the basis for future clinical studies examining therapeutic efficacy in a preselected patient population with GOF TP53 mutations.

The homologous recombination repair (HRR) pathway is required for high-fidelity repair of double-strand DNA breaks. Cancer cells with HRR deficiency due to somatic BRCA1 or BRCA2 mutations are sensitive to platinum agents and targeted therapy with PARP inhibitors. Endometrial cancer is the most common cancer of the female reproductive system. DNA damage agents are commonly used in treatment but tumors often develop resistance. Metadherin (MTDH) has been shown to be amplified in a wide range of solid tumors as well as in leukemia and lymphoma. Overexpression of MTDH is correlated with angiogenesis, metastasis, and chemoresistance. Histone lysine N-methyl-transferase G9a catalyzes the demethylation of histone H3 at lysine residue 9 (H3K9) resulting in a repressive histone modification. The objective of this study is to validate the function of MTDH and G9a in HRR and test the potential to target MTDH or G9a to increase drug sensitivity. p21 expression was induced when G9a was depleted. DNA damage induced MTDH expression at 4 hours after 8Gy γ-irradiation in the well validated endometrial adenocarcinoma cell line Ishikawa H. However, induction of MTDH protein expression by the same radiation dosage was reduced when G9a was depleted. The G9a inhibitor UNC0642 itself can also induce the expression of MTDH after 24 hours treatment. Ishikawa parental cells and two Ishikawa sub-cell lines in which G9a was depleted by CRISPR were tested for the DNA damage biomarker γH2Ax and Rad51 foci formation at 4 hours following 8Gy irradiation. G9a depletion was shown to decrease Rad51 foci formation and to increase the formation of γH2Ax foci. Two natural products, Celastrol and Pristimerim were found to inhibit MTDH, FANCD2 and FANCI protein levels, which may be used to induce BRCA-ness in homologous recombination repair proficient cancer cells. G9a depletion results in the reduction of irradiation-induced Rad51 foci formation and an increase of irradiation-induced γH2Ax foci formation. We suggest that G9a depletion can increase cancer cell sensitivity to DNA damage agents. MTDH is induced by DNA damage and is a G9a inhibitor. MTDH depletion causes reduction of irradiation-induced Rad51 foci. This study is supported by the University of Iowa Department of Obstetrics and Gynecology Research Development Fund, NIH grant R01CA99908 and R01CA184101. Citation Format: Xiangbing Meng, Shujie Yang, Jianling Bi, Yiyang Li, Yuping Zhang, Sudartip Areecheewaku, Mary Li, Zihan Wang, Aliasger K. Salem, Kimberly K. Leslie. Induction of HRD by targeting G9a and MTDH in DNA repair proficient endometrial cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4852.